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Biomedical subjects

A Berns

Publications and source records attributed to A Berns.

At least 73 records · Page 4Linked to original sources
PubMed1995

Transformation of axial skeleton due to overexpression of bmi-1 in transgenic mice.

The oncogene bmi-1, which was originally found to be involved in B- and T-cell lymphoma formation encodes a protein with a domain of homology to the Drosophila protein Posterior sex combs (Psc) and its relative Suppressor 2 of Zeste (Su(z)2) (refs 4 and 5). Psc is a member of the Polycomb-group gene family, which is required to maintain the repression of homeotic genes that regulate the identities of Drosophila segments. The possibility that bmi-1 may play a similar role in vertebrates was suggested by our previous finding that mice lacking the bmi-1 gene show posterior transformations of the axial skeleton. Here we report that transgenic mice overexpressing Bmi-1 protein show the opposite phenotype, namely a dose-dependent anterior transformation of vertebral identity. The anterior expression boundary of the Hoxc-5 gene is shifted in the posterior direction, indicating that Bmi-1 is involved in the repression of Hox genes. We propose that Bmi-1 is a member of a vertebrate Polycomb complex that regulates segmental identity by repressing Hox genes throughout development.

Animals↗
PubMed1995

Gene therapy for hemophilia A: production of therapeutic levels of human factor VIII in vivo in mice.

Continuous delivery of factor VIII (FVIII) protein in hemophiliacs by gene therapy will represent a major clinical advance over the current practice of infrequent administration of purified FVIII. Conceptually, retroviral vectors that can permanently insert the FVIII gene into the DNA of the host cell appear the most suitable vehicles for this specific purpose. However, most retroviral vector systems have shown a poor performance in the production of FVIII from primary cells in vitro and in vivo. Here we report the retroviral-mediated gene delivery of a B-domain-deleted human FVIII by using the MFG vector system. This vector permitted efficient transduction of the majority of the primary cells in culture without the use of a selectable marker. High levels of FVIII were produced by various transduced primary cells in vitro. Upon transplantation of primary fibroblasts into mice, therapeutic levels of FVIII in the circulation were obtained for > 1 week. The capacity of primary cells to deliver the FVIII into the circulation was strongly dependent on the site of implantation. These results represent a major step forward in development of gene therapy for treating hemophilia A.

3T3 Cells↗
PubMed1995

Increase in positive selection of CD8+ T cells in TAP1-mutant mice by human beta 2-microglobulin transgene.

Mice harboring a deletion of the gene encoding the transporter associated with antigen presentation-1 (TAP1) are impaired in providing major histocompatibility complex (MHC) class I molecules with peptides of cytosolic origin and lack stable MHC class I cell surface expression. They consequently have a strongly reduced number of CD8+ T cells. To examine whether selection of CD8+ T cells is dependent on TAP-dependent peptides, we partially restored MHC class I cell surface expression in TAP1-deficient mice by introduction of human beta 2-microglobulin. We show that selection of functional CD8+ T cells can be augmented in vivo in the absence of TAP1-dependent peptides.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Adenosine-deaminase-deficient mice die perinatally and exhibit liver-cell degeneration, atelectasis and small intestinal cell death.

We report the generation and characterization of mice lacking adenosine deaminase (ADA). In humans, absence of ADA causes severe combined immunodeficiency. In contrast, ADA-deficient mice die perinatally with marked liver-cell degeneration, but lack abnormalities in the thymus. The ADA substrates, adenosine and deoxyadenosine, are increased in ADA-deficient mice. Adenine deoxyribonucleotides are only modestly elevated, whereas S-adenosylhomocysteine hydrolase activity is reduced more than 85%. Consequently, the ratio of S-adenosylhomocysteine (AdoMet) to S-adenosyl homocysteine (AdoHcy) is reduced threefold in liver. We conclude that ADA plays a more critical role in murine than human fetal development. The murine liver pathology may be due to AdoHcy-mediated inhibition of AdoMet-dependent transmethylation reactions.

Adenosine Deaminase↗
PubMed1995

Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth.

B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons.

Animals↗
PubMed1994

Developmental rescue of an embryonic-lethal mutation in the retinoblastoma gene in chimeric mice.

The requirement for a functional retinoblastoma gene, Rb-1, in murine development around days 12-15 of gestation precludes monitoring the effect of loss of Rb-1 function on later stages of development and on tumorigenesis in adult mice. Here we describe the developmental rescue of embryonic stem cells carrying two inactive Rb-1 alleles in chimeric mice. Rb-1- cells contributed substantially to most tissues in adult chimeras, including blood, liver and central nervous system, which were severely affected in pure Rb-1- embryos. The adult chimeric erythroid compartment appeared completely normal, but an increased number of nucleated red cells was observed during fetal liver erythropoiesis in highly chimeric embryos. No ostensive abnormalities were seen in the developing and adult CNS. However, the developing retina of chimeric Rb-1- embryos showed ectopic mitoses and substantial cell degeneration, while the contribution of Rb-1- cells to the adult retina was much reduced. Moreover, the formation of lens fibre cells was severely disturbed. No retinoblastomas developed in any of these mice. Instead, nearly all animals died of pituitary gland tumours which were exclusively derived from Rb-1- cells.

Animals↗
PubMed1994

Identification of an invasion-inducing gene, Tiam-1, that encodes a protein with homology to GDP-GTP exchangers for Rho-like proteins.

Using proviral tagging in combination with in vitro selection for invasiveness, we have identified a gene, designated Tiam-1, that affects invasion. In the selected invasive T lymphoma variants, proviral insertions were found within coding exons of the Tiam-1 gene, resulting in both truncated 5'-end and 3'-end transcripts that give rise to N- and C-terminal Tiam-1 protein fragments. In one invasive variant, amplification of the Tiam-1 locus was observed with concomitant increase in the amount of normal Tiam-1 protein. Cell clones that were invasive in vitro produced experimental metastases in nude mice, and transfection of truncated Tiam-1 cDNAs into noninvasive cells made these cells invasive. The predicted Tiam-1 protein harbors a Dbl- and Pleckstrin-homologous domain, which it shares with GDP-GTP exchangers for Rho-like proteins that have been implicated in cytoskeletal organization.

Amino Acid Sequence↗
PubMed1994

CD3 components at the surface of pro-T cells can mediate pre-T cell development in vivo.

Developmentally arrested pro-T cells (CD4-8-, IL-2R+, HSA++) of RAG-1-deficient mice appear to express low levels of CD3 molecules in the absence of T cell receptor (TcR) chains at their surface, while developmentally arrested pre-T cells of TcR alpha-deficient mice express low levels of a disulfide-linked TcR beta chain in association with CD3 molecules. Cross-linking of the CD3 modules on pro-T cells of RAG-1-/- mice in vivo, with either of two different CD3 epsilon-specific monoclonal antibodies, induces differentiation of these pro-T cells into pre-T cells (CD4+8+, IL-2R-, HSA+), concomitant with a rapid expansion of the thymic T cell compartment, up to 175-fold within 12 days. The same effects can be produced by introduction of a mutant TcR beta transgene lacking most of the variable domain (delta V-TcR beta) into the RAG-1-/- background. These experiments suggest that cross-linking of the CD3 modules on pro-T cells mimics the signaling function expected of the pre-TcR complex, which is found at the surface of pre-T cells prior to functional TcR alpha gene rearrangement. The variable domain of the TcR beta chain is apparently not essential for inducing these aspects of T cell development.

Animals↗
PubMed1994

Thymic lymphomas in interleukin 9 transgenic mice.

Transgenic mice overexpressing the interleukin 9 gene were generated to study the biological activity of this cytokine in vivo. Although no major histological or morphological modifications of the lymphoid system were observed in most animals, approximately 7% of transgenic mice developed thymic lymphomas at the age of 3-9 months. The tumor cells, which were clonal, with unique T cell rearrangements, were double positive for the expression of CD4 and CD8. The need for additional transforming events, suggested by the low incidence of spontaneous tumors, was further indicated by the high susceptibility of the transgenic animals to injections of low doses of N-methyl-N-nitrosourea, a chemical carcinogen with a thymic tropism. Expression of interleukin 9 was required for optimal tumor growth in vivo, as one of the tumors studied, which had lost the transgene, was much more efficiently transplanted into transgenic than in normal mice. Moreover, the in vitro proliferative activity of interleukin 9 on cell lines derived from such transgene-negative tumors suggests that an autocrine loop mediates the proliferation of these cells in vivo. Taken together, these results indicate that dysregulated IL-9 expression could be involved in the development of some T cell malignancies.

Animals↗
PubMed1993

Pim-1 levels determine the size of early B lymphoid compartments in bone marrow.

The mouse proto-oncogene Pim-1, which encodes two cytoplasmic serine-threonine-specific protein kinases, is frequently activated by proviral insertion in murine leukemia virus-induced hematopoietic tumors. Transgenic mice overexpressing Pim-1 show a low incidence of spontaneous T cell lymphomas, whereas null mutant mice lack an obvious phenotype. We have analyzed the early B lymphoid compartment from both null mutant and E mu-Pim-1 transgenic mice. The level of Pim-1 expression appears to be a determining factor in the ability of these cells to respond to the growth factors interleukin 7 (IL-7) and SF (steel factor). The impaired response in null mutant mice could be rescued by introduction of a functional Pim-1 transgene. Moreover, overexpression of Pim-1 facilitates the derivation of primitive lymphoid cell lines that are dependent on combined stimulation with IL-7 and SF or insulin-like growth factor 1. These results for the first time identify the involvement of Pim-1 in a normal cellular function, as an important regulator of early B lymphopoiesis in mice.

Animals↗
PubMed1993

In vivo analysis of Pim-1 deficiency.

The Pim-1 proto-oncogene encodes a highly conserved serine/threonine phosphokinase which is predominantly expressed in hematopoietic organs and gonads in mammals. Overexpression of Pim-1 predisposes to lymphomagenesis in mice. To develop a further understanding of Pim-1 in molecular terms, as well as in terms of its potential role in hematopoietic development, we have generated mice deficient in Pim-1 function. Pim-1-deficient mice are ostensibly normal, healthy and fertile. Detailed comparative analyses of the hematopoietic systems of the mutant mice and their wild-type littermates showed that they are indistinguishable for most of the parameters studied. Our analyses revealed one unexpected phenotype that correlated with the level of Pim-1 expression: Pim-1 deficiency correlated with a erythrocyte microcytosis, whereas overexpression of Pim-1 in E mu-Pim-1-transgenic mice resulted in erythrocyte macrocytosis. In order to confirm that the observed decrease in erythrocyte Mean Cell Volume (MCV) was attributable to the Pim-1 deficiency, we developed mice transgenic for a Pim-1 gene construct with its own promoter and showed that this transgene could restore the low erythrocyte Mean Cell Volume observed in the Pim-1-deficient mice to near wild-type levels. These results might be relevant to the observed involvement of the Pim-1 gene in mouse erythroleukemogenesis. The surprising lack of a readily observed phenotype in the lymphoid compartment of the Pim-1-deficient mice, suggests a heretofore unrecognized degree of in vivo functional redundancy of this highly conserved proto-oncogene.

Animals↗
PubMed1993

Impaired interleukin-3 response in Pim-1-deficient bone marrow-derived mast cells.

The mouse Pim-1 gene encodes two cytoplasmic serine-threonine-specific protein kinases. The gene has been found to be activated (overexpressed) by retroviral insertion in hematopoietic tumors in mice. Transgenic mice that overexpress Pim-1 (E mu-Pim-1) have a low incidence of spontaneous T-cell lymphomas and an increased susceptibility to Moloney murine leukemia virus and N-ethyl-N-nitrosourea-induced lymphomas. Apart from a slight enlargement of the spleen, no abnormalities were found in prelymphomatous transgenic mice. Inactivation of the Pim-1 gene in the germline of mice resulted in mice with a surprisingly subtle phenotype. Therefore, we investigated whether subtle effects of the absence of Pim-1 could be made visible during in vitro culturing of hematopoietic cells. We found that bone marrow-derived mast cells (BMMC) lacking Pim-1 had a distinct growth disadvantage when grown on interleukin (IL)-3, but not when stimulated by the factors IL-4, IL-9, or Steel factor (SF). This indicates a role for Pim-1 as a modulator of the IL-3 signal transduction pathway.

Animals↗
PubMed1993

Characterization and chromosomal localization of the human proto-oncogene BMI-1.

The proto-oncogene bmi-1 is frequently activated by Moloney murine leukemia proviral insertions in E mu-myc transgenic mice1,2. Using a mouse bmi-1 cDNA probe a transcript of 3.3 kb was detected on Northern blots of human Burkitt's lymphoma cell lines. We have isolated and sequenced cDNA clones from a human erythroleukemia cell line (K562) derived cDNA library, using different mouse bmi-1 cDNA fragments as a probe. Analysis of genomic BMI-1 sequences reveals a gene structure which is very similar to that of the mouse, consisting of at least 10 exons. The human cDNA is 3203 bp in length and shows 86% identity to the mouse nucleotide sequence. The open reading frame encodes a protein of 326 amino acids which shares 98% identity to the amino acid sequence of mouse bmi-1 protein. In vitro translation experiments show that human cDNA derived RNA translates into a protein with a mobility of 44-46 kD on SDS polyacrylamide gels. Fluorescence in situ hybridization (FISH) on metaphase chromosome spreads located the human BMI-1 gene to the short arm of chromosome 10 (10p13), a region known to be involved in translocations in various leukemias.

Amino Acid Sequence↗
PubMed1993

Ribonucleotide reductase-deficient mutants of pseudorabies virus are avirulent for pigs and induce partial protective immunity.

We have mutagenized and mapped the gene encoding the large subunit of ribonucleotide reductase (RR1) in pseudorabies virus (PRV; synonyms Aujeszky's disease virus, suid herpesvirus type 1). PRV strains carrying an oligonucleotide that leads to termination of translation of the RR1 gene are avirulent for mice. We subsequently constructed a PRV strain carrying a deletion in the RR1 gene and also a PRV strain carrying both the deletion in the RR1 gene and a deletion in the glycoprotein g1 gene, which is a marker for PRV virulence. Both PRV strains were assayed for virulence and immunogenicity in pigs, the natural host for PRV. In contrast to a marker-rescued PRV strain, these RR1-deleted mutants were avirulent, were shed in very low titres in the oropharyngeal fluid by the animals, and induced low titres of neutralizing antibodies. However, protection against clinical signs after infection with virulent PRV was induced by both RR1-deleted mutants. The relative importance of viral RR and thymidine kinase enzymes for deoxynucleotide synthesis in viral replication is discussed. In addition, we discuss the potential use of RR as a target for anti-herpesviral drugs and the use of PRV strains, deleted for the RR1 gene, as vaccine strains.

Amino Acid Sequence↗
PubMed1993

Analysis of Pim-1 function in mutant mice.

The Pim-1 gene has frequently been found activated by proviral insertion in haematopoietic tumors in mice. The fact that overexpression of Pim-1 can contribute to lymphomagenesis was formally proven by overexpressing a Pim-1 transgene in lymphoid cells. The transgene induces a low incidence of T cell lymphomas and an increased susceptibility to chemically (ENU) and virally (MoMuLV) induced lymphomas. The mouse Pim-1 gene encodes two cytoplasmic protein-serine/threonine kinases. Northern analysis shows the highest expression to be in haematopoietic tissues, especially early in development. High expression has also been noted in testis and ES cells. Expression can be induced by growth factors and mitogens. The gene is evolutionarily highly conserved. Inactivation of both Pim-1 alleles in ES cells or mice did not reveal any obvious abnormalities. In order to look more closely for possible haematopoietic abnormalities specific growth factor response were studied in vitro. The IL-3 response of bone marrow-derived mast-cell cultures (BMMC) was found to be severely impaired in mast cells derived from Pim-1 deficient mice.

Alleles↗
PubMed1992

Requirement for a functional Rb-1 gene in murine development.

Human retinoblastomas can occur both as hereditary and as sporadic cases. Knudson's proposal that they result from two mutational events, of which one is present in the germ line in hereditary cases, has been confirmed by more recent molecular analysis, which has shown both events to involve loss or mutational inactivation of the same gene, RB-1 (ref. 2). RB-1 heterozygosity also predisposes to osteosarcoma, and RB-1 allele losses are seen in sporadic lung, breast, prostate and bladder carcinomas. RB-1 is expressed in most, if not all, tissues and codes for a nuclear phosphoprotein which becomes hypophosphorylated in the G0 growth arrest state and in the G1 phase of the cell cycle. To gain a further insight into the role of RB-1 we and other groups have generated mice carrying an inactivated allele of the homologous gene, Rb-1 (ref. 10), by gene targeting. We report here that young heterozygous mice do not appear abnormal and do not develop retinoblastoma at a detectable frequency. However, homozygous mutant embryos fail to reach term and show a number of abnormalities in neural and haematopoietic development. Broadly similar results are reported by the other groups.

Abnormalities, Multiple↗