Consequences of Postnatally Elevated Insulin-Like Growth Factor-II in Transgenic Mice: Endocrine Changes and Effects on Body and Organ Growth.

Insulin-like growth factor-II (IGF-II) is an important regulator of embryonic growth and differentiation, but its function in postnatal life is unclear. To address this point, we generated transgenic mice harboring fusion genes in which a human IGF-II complementary DNA is placed under the transcriptional control of the rat phosphoenolpyruvate carboxykinase promoter. Transgene-specific messenger RNA was detected in liver, kidney, and several parts of the gut. Serum IGF-II levels in transgenic mice were 2-3 times higher than those in controls and increased after starvation. Circulating IGF-I correlated negatively and IGF-binding protein-2 (IGFBP-2) positively with IGF-II levels, suggesting that IGF-I is displaced from IGFBPs by IGF-II and that IGFII is a major regulator of IGFBP-2. Serum levels of IGFBP-3 and IGFBP-4 tended to be higher in phosphoenolpyruvate carboxykinase- IGF-II transgenic mice than in controls, as evaluated by ligand blot analysis. Starvation reduced serum IGF-I, but increased IGFBP-2 in transgenic mice more markedly than in controls. Fasting insulin levels were significantly reduced in transgenic mice, whereas glucose levels were not influenced by elevated IGF-II. The body growth of 4- and 12- week-old mice was not significantly influenced by elevated IGF-II, but transgenic mice displayed increased kidney and testis weight at the age of 4 weeks, and increased adrenal weight at the age of 12 weeks. Our results demonstrate that elevated IGF-II in postnatal life has multiple endocrine consequences and subtle time-specific effects on organ growth

Establishment of pluripotent cell lines from vertebrate species - Present status and future prospects

Pluripotent embryonic stem (ES) cells are undifferentiated cell lines derived from early embryos and are capable of unlimited undifferentiated proliferation in vitro. They retain the ability to differentiate into all cell types including germ cells in chimeric animals in vivo, and can be induced to form derivatives of all three germ layers in vitro. Mouse ES cells represent one of the most important tools in genetic research. Major applications include the targeted mutation of specific genes by homologous recombination and the discovery of new genes by gene trap strategies. These applications would be of high interest for other model organisms and also for livestock species, However, in spite of tremendous research activities, no proven ES cells colonizing the germ line have been established for vertebrate species other than mouse a nd chicken thus far. This review summarizes the current status of deriving pluripotent embryonic stem cell lines from vertebrates and recent developments in nuclear transfer technology, which may provide an alternative tool for genetic modification of livestock animals. Copyright (C) 1999 S. Karger AG, Basel

Charakterisierung der Resistenz von Winterweizensorten gegenüber Steinbrand (Tilletia caries)

Attack with common bunt (Tilletia caries) is a potential threat to wheat production in organic farming. The situation may even become worse if (in agreement with EU-legislation) only organically produced seed is used in organic farming. The targeted use of resistant varieties could be a way to alleviate the problem. However, knowledge regarding bunt resistance of wheat varieties on the one hand and aggressiveness in populations of the bunt pathogen on the other hand is still insufficient. In the frame of a federal research programme for organic farming (BÖL),30 winter wheat varieties and breeding lines were studied at five different locations using spores of local origin in order to assess the natural level of resistance (WÄCHTER et al. 2004). In addition, the different spore accessions were analysed with a set of differential varieties supplied by B. Goates (USDA-ARS, Aberdeen, ID). Since the impact of bunt is only obvious after ear appearance, utilization of early (leaf) symptoms (KOCH & SPIESS, 2002) and immunological detection of T. caries in planta with an enzyme-linked immunosorbent assay (ELISA) (EIBEL 2002) were evaluated as diagnostic tools to characterise the level of resistance

Pairwise disjoint perfect matchings in rr-edge-connected rr-regular graphs

Thomassen [Problem 1 in Factorizing regular graphs, J. Combin. Theory Ser. B, 141 (2020), 343-351] asked whether every $r$-edge-connected $r$-regular graph of even order has $r-2$ pairwise disjoint perfect matchings. We show that this is not the case if $r \equiv 2 \text{ mod } 4$. Together with a recent result of Mattiolo and Steffen [Highly edge-connected regular graphs without large factorizable subgraphs, J. Graph Theory, 99 (2022), 107-116] this solves Thomassen's problem for all even $r$. It turns out that our methods are limited to the even case of Thomassen's problem. We then prove some equivalences of statements on pairwise disjoint perfect matchings in highly edge-connected regular graphs, where the perfect matchings contain or avoid fixed sets of edges. Based on these results we relate statements on pairwise disjoint perfect matchings of 5-edge-connected 5-regular graphs to well-known conjectures for cubic graphs, such as the Fan-Raspaud Conjecture, the Berge-Fulkerson Conjecture and the $5$-Cycle Double Cover Conjecture.Comment: 24 page

Growth hormone (GH)-transgenic insulin-like growth factor 1 (IGF1)-deficient mice allow dissociation of excess GH and IGF1 effects on glomerular and tubular growth

Growth hormone (GH)-transgenic mice with permanently elevated systemic levels of GH and insulin-like growth factor 1 (IGF1) reproducibly develop renal and glomerular hypertrophy and subsequent progressive glomerulosclerosis, finally leading to terminal renal failure. To dissociate IGF1-dependent and -independent effects of GH excess on renal growth and lesion development in vivo, the kidneys of 75 days old IGF1-deficient (I-/-) and of IGF1-deficient GH-transgenic mice (I-/-/G), as well as of GH-transgenic (G) and nontransgenic wild-type control mice (I+/+) were examined by quantitative stereological and functional analyses. Both G and I-/-/G mice developed glomerular hypertrophy, hyperplasia of glomerular mesangial and endothelial cells, podocyte hypertrophy and foot process effacement, albuminuria, and glomerulosclerosis. However, I-/-/G mice exhibited less severe glomerular alterations, as compared to G mice. Compared to I+/+ mice, G mice exhibited renal hypertrophy with a significant increase in the number without a change in the size of proximal tubular epithelial (PTE) cells. In contrast, I-/-/G mice did not display significant PTE cell hyperplasia, as compared to I-/- mice. These findings indicate that GH excess stimulates glomerular growth and induces lesions progressing to glomerulosclerosis in the absence of IGF1. In contrast, IGF1 represents an important mediator of GH-dependent proximal tubular growth in GH-transgenic mice

Insulin-like growth factor-binding protein-2 inhibits proliferation of human embryonic kidney fibroblasts and of IGF-responsive colon carcinoma cell lines

AbstractSo far, the physiological role of insulin-like growth factor binding protein-2 (IGFBP-2) has not been demonstrated directly. Therefore, we transfected 293 cells with an expression vector containing the CMV promoter and the complete cDNA of mouse IGFBP-2. Secretion of bioactive IGFBP-2 into conditioned medium was demonstrated by Western ligand and Western immunoblotting and quantified by specific RIA. For the analysis of cell proliferation three clones exhibiting either high or low/no IGFBP-2 expression were selected and compared to non-transfected parental 293 cells. IGFBP-2 secreting clones displayed reduced conversion of thiazolyl blue when compared to negative clones or non-transfected parental 293 cells (P<0.01). The lower growth activity measured in the IGFBP-2 secreting clones was compensated in great part by the administration of exogenous IGF-I or -II. Conditioned media of IGFBP-2 secreting clones inhibited growth of IGF-responsive colon tumor cell lines (LS513, HT-29) while those of negative clones did not. In addition, conditioned medium from a clone expressing high levels of IGFBP-2 inhibited anchorage-independent growth of LS513 and HT-29 cells. In contrast, growth of an IGF-unresponsive tumor cell line (Co-115) was not affected by the conditioned media. We hypothesize that IGFBP-2 might sequester the IGFs and thus prevent them from transferring their mitogenic signals

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background: Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass. Results: Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa. Conclusions: Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa

3D structured illumination microscopy of mammalian embryos and spermatozoa

Background: Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) is well established on flat, adherent cells. However, blastomeres of mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to the movement during scanning and the large distance to the cover glass. Results: Here we present a highly detailed protocol that allows performing 3D-SIM on blastomeres of mammalian embryos with an image quality comparable to scans in adherent cells. This protocol was successfully tested on mouse, rabbit and cattle embryos and on rabbit spermatozoa. Conclusions: Our protocol provides detailed instructions on embryo staining, blastomere isolation, blastomere attachment, embedding, correct oil predictions, scanning conditions, and oil correction choices after the first scan. Finally, the most common problems are documented and solutions are suggested. To our knowledge, this protocol presents for the first time a highly detailed and practical way to perform 3D-SIM on mammalian embryos and spermatozoa