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Biomedical subjects

A Bernstein

Publications and source records attributed to A Bernstein.

At least 109 records · Page 6Linked to original sources
PubMed1992

Retroviral insertions downstream of the heterogeneous nuclear ribonucleoprotein A1 gene in erythroleukemia cells: evidence that A1 is not essential for cell growth.

A large number of novel cellular proto-oncogenes have been identified and cloned by analysis of common integration sites in retrovirally induced malignancies. In the multistage erythroleukemias induced by the various strains of Friend leukemia virus, the analysis of proviral-integration events has led to the identification of two genes, Fli-1 and Spi-1, both novel members of the ets oncogene family of transcription factors. In this report, we describe the identification of another integration site, designated Fli-2 (Friend leukemia virus integration-2), in an erythroleukemia cell line induced by Friend murine leukemia virus (F-MuLV). Rearrangements at the Fli-2 locus were found in two erythroleukemia cell lines independently induced by F-MuLV and one leukemic cell line derived from the spleen of a mouse infected with the polycythemia strain of Friend leukemia virus. The deduced amino acid sequence of a cDNA corresponding to a transcript originating from genomic DNA adjacent to Fli-2 is identical to that of the human heterogeneous nuclear ribonucleoprotein A1 gene, a member of the gene family of RNA-binding proteins involved in RNA splicing. In one erythroleukemia cell line, A1 expression was undetectable as a result of F-MuLV integration in one allele and loss of the other allele. These results suggest that perturbations in RNA splicing mechanisms may contribute to malignant transformation and provide direct evidence that the A1 protein is not required for cell growth.

Amino Acid Sequence↗
PubMed1992

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors.

Animals↗
PubMed1992

Embryology and morphology of microphthalmia in transgenic mice expressing a gamma F-crystallin/diphtheria toxin A hybrid gene.

BACKGROUND: Transgenic mice in which elongating lens fiber cells were ablated resulting in microphthalmia have been reported, however, their embryology and detailed morphology have not. EXPERIMENTAL DESIGN: The morphology of homozygous and hemizygous CDI, transgenic mice carrying the gamma FDT-A gene was studied by light microscopy on different days of gestation as well as postpartum. The findings were compared with normal CD-1 wild type controls. RESULTS: The earliest changes in mouse embryos transgenic for the gamma F-crystallin/diphtheria toxin A transgene are seen on day 12, when apoptotic cells appear in the area of elongation. In hemizygous embryos, ocular development is relatively normal until day 17 when the lens and eye are slightly smaller than normal and the lens vesicle is filled with abnormal lens material. At this time, the posterior capsule of the lens may rupture, releasing abnormal lens material which disperses throughout the eye, perturbing growth and other ocular structures. Additional breaks may subsequently occur and the ultimate morphology of the hemizygotes correlates with when the posterior capsule ruptures, how much lens material is released, and where it disperses. In homozygous embryos, due to extensive ablation of lens fiber cells, the "lens" becomes a diminutive mass of abnormal lens material, posteriorly located within the eye, and otherwise unable to fulfill its mechanical or inductive role in the development of the cornea, anterior chamber, iris, ciliary epithelium, and zonules with the result that all of these structures are markedly abnormal or absent. In addition, the lens is necessary for the accumulation of vitreous which in turn is required for the growth of the eye as a whole. In homozygous animals, vitreous does not accumulate and severe microphthalmia results. CONCLUSIONS: This study confirms and extends previous observations and conclusions on the central, orchestrating role of the lens in the development of the eye and illustrates the power of transgenic technology to elucidate the finer points of mammalian ocular development.

Animals↗
PubMed1991

Diamond-Blackfan anemia: heterogenous response of hematopoietic progenitor cells in vitro to the protein product of the steel locus.

Diamond-Blackfan anemia is a congenital disorder of erythropoiesis in humans, characterized by a macrocytic anemia often associated with physical anomalies. Mutations at either the W or Steel loci in the mouse also leads to a severe macrocytic anemia, as well as other developmental abnormalities. The W locus encodes the proto-oncogene c-kit, a member of the receptor tyrosine kinase family, while the Steel locus encodes a potent hematopoietic growth factor that is the ligand for c-kit. Growth of clonogenic marrow erythroid progenitor cells in vitro in the presence of the recombinant hematopoietic growth factors interleukin-3 (IL-3) and Steel was used to characterize this disease at the cellular level. Three patterns of in vitro marrow response to both recombinant IL-3 or Steel were observed among 10 Diamond-Blackfan patients: those that responded quantitatively and qualitatively almost as well as cells from normal marrow, those that responded at an intermediate level, and those that did not respond at all. These results provide evidence for cellular heterogeneity underlying the pathogenesis of this disorder and therefore raise the possibility that there may be more than one underlying molecular basis for the disease. No gross abnormalities in the structure of either the c-kit or Steel loci were observed in these patients. The normal response in culture of the progenitor cells from at least some patients to Steel with or without IL-3 raises the possibility of using this novel growth factor as a therapeutic agent in Diamond-Blackfan anemia.

Adolescent↗
PubMed1991

Long-term reconstitution of the mouse hematopoietic system by embryonic stem cell-derived fetal liver.

Murine embryonic stem (ES) cells are permanent blastocyst-derived cell lines capable of contributing to a wide variety of tissues, including the germ line, after injection into host blastocysts. Recently, we have shown that ES cells can produce all of the cells of the developing fetus after aggregation with developmentally compromised tetraploid embryos. Completely ES cell-derived embryos die perinatally, but the liver of these embryos is a source of entirely ES cell-derived hematopoietic progenitors. We have taken 14- to 15-day fetal liver cells from ES cell-tetraploid chimeras and reconstituted the hematopoietic system of lethally irradiated adult recipient mice. ES cell-derived hematopoietic stem cells were capable of long-term (greater than 6 months) repopulation of irradiated recipients, and all hematopoietic cell lineages analyzed (erythrocytes, T cells, mast cells, and macrophages) were derived exclusively from ES cells in such recipients. Thus, ES cells retain the capacity to differentiate into all hematopoietic cell types after prolonged passage in culture. This approach should provide a direct route to the production of mice whose hematopoietic cells carry genetic alterations that would be lethal if passed through the germ line.

Animals↗
PubMed1991

The c-fms gene complements the mitogenic defect in mast cells derived from mutant W mice but not mi (microphthalmia) mice.

Mutations at three loci in the mouse--W, Steel Sl), and microphthalmia (mi)--can lead to a deficiency in melanocytes and mast cells. As well, W and Sl mutants can be anemic and sterile, whereas mi mice are osteopetrotic due to a monocyte/macrophage defect. Recent data have shown that the c-kit receptor tyrosine kinase is the gene product of the W locus, whereas Sl encodes the ligand for this growth factor receptor. We show here that ectopic expression of c-fms, a gene that encodes a macrophage growth factor receptor that is closely related to the c-kit receptor, complements mutations at the W locus in an in vitro mast cell/fibroblast coculture system but is unable to reverse the inability of mi/mi mast cells to survive under these conditions. Furthermore, mast cells expressing the c-fms receptor survive on a monolayer of fibroblasts homozygous for the Sl mutation. These results suggest that ligand binding to the c-kit or c-fms receptor activates identical or overlapping signal transduction pathways. Furthermore, they suggest that mi encodes a protein necessary for transducing signals mediated by way of either the c-kit or c-fms receptor.

Animals↗
PubMed1991

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

A mammalian protein kinase with potential for serine/threonine and tyrosine phosphorylation is related to cell cycle regulators.

In a screen of mouse erythroleukemia cDNA expression libraries with anti-phosphotyrosine antibodies, designed to isolate tyrosine kinase coding sequences, we identified several cDNAs encoding proteins identical or very similar to known protein-tyrosine kinases. However, two frequently isolated cDNAs, clk and nek, encode proteins which are most closely related to protein kinases involved in regulating progression through the cell cycle, and contain motifs generally considered diagnostic of protein-serine/threonine kinases. The clk gene product contains a C-terminal cdc2-like kinase domain, most similar to the FUS3 catalytic domain. The Clk protein, expressed in bacteria, becomes efficiently phosphorylated in vitro on tyrosine as well as serine/threonine, and phosphorylates the exogenous substrate poly(glu, tyr) on tyrosine. Direct biochemical evidence indicates that both protein-tyrosine and protein-serine/threonine kinase activities are intrinsic to the Clk catalytic domain. These results suggest the existence of a novel class of protein kinases, with an unusual substrate specificity, which may be involved in cell cycle control.

Amino Acid Sequence↗
PubMed1991

Purification and characterization of a rabbit salivary protein, a potent inhibitor of crystal growth of calcium phosphate salts.

Human saliva is supersaturated with respect to basic calcium phosphate salts but is stabilized by specific macromolecules that inhibit calcium phosphate precipitation. One of the families of inhibitory proteins in human and monkey saliva is the acidic proline-rich proteins. The purpose of this study was to isolate and characterize inhibitors of calcium phosphate precipitation from rabbit parotid saliva. Saliva was fractionated by immunoaffinity chromatography and anion exchange chromatography. Individual fractions were assayed for their ability to inhibit calcium phosphate crystal growth and the fraction associated with the inhibition was purified by repeated anion exchange chromatography, preparative gel electrophoresis and electroelution. A major (APRP) and two minor proteins (AM1, AM2) that were inhibitory were purified. APRP is an acidic proline-rich phospho-glycoprotein and a very potent inhibitor of secondary crystal growth of calcium phosphate as it was active at a concentration of 2 x 10(-8) M in a standard assay. The N-terminal sequence of one APRP was EYENLDGSLAATQNDDD?Q and a clostripain fragment of APRP had the following N-terminal sequence PQHRPPRPGGH-????SPPP?GN???PPP. Although the N-terminal segment of APRP does not resemble that of proline-rich proteins, alignment of the clostripain fragment with the repeat region of such proteins from rat, mouse, monkey and man revealed a high degree of similarity, indicating a structural relationship with the proline-rich protein family.

Amino Acid Sequence↗
PubMed1991

Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.

The retroviral integration site Fli-1 is rearranged in 75% of the erythroleukemia cell clones induced by Friend murine leukemia virus (F-MuLV), whereas Spi-1/PU.1, a member of the ets family of DNA-binding proteins, is rearranged in 95% of the erythroleukemias induced by Friend spleen focus-forming virus (SFFV). To determine the transcriptional domain defined by Fli-1, we have isolated a cDNA clone that is highly expressed only in erythroleukemia cell lines with Fli-1 rearrangements. The protein sequence of this cDNA is very similar to Erg2, another member of the ets gene family. The hydrophilic carboxy-terminal end of the Fli-1 cDNA shares significant sequence similarity to the DNA-binding ETS domain found in all members of the ets family. PFGE analysis localized Fli-1 within 240 kb of the ets-1 proto-oncogene on mouse chromosome 9 and human chromosome 11q23, suggesting that ets-1 and Fli-1 arose from a common ancestral gene by gene duplication. The involvement of the murine Fli-1, Spi-1, and avian v-ets genes in erythroleukemia induction suggests that activation of ets gene family members plays an important role in the progression of these multistage malignancies.

Amino Acid Sequence↗
PubMed1991

Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression.

Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, remained sensitive to HIV-1 infection. In contrast, resistance to HIV-1 infection was observed in cells expressing antisense RNA to the HIV-1 primer-binding site or to the region 5' to the primer-binding site as part of the 3' region of the npt mRNA. Cells expressing the tat mRNA leader sequence in the sense orientation as a precise replacement of the 5' untranslated region of npt mRNA were also resistant to HIV-1. These results indicate that sense and antisense approaches can be used to interfere with HIV-1 multiplication.

Base Sequence↗
PubMed1991

The Steel/W transduction pathway: kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor.

The W/c-kit and Steel loci respectively encode a receptor tyrosine kinase (Kit) and its extracellular ligand, Steel factor, which are essential for the development of hematopoietic, melanocyte, and germ cell lineages in the mouse. To determine the biochemical basis of the Steel/W developmental pathway, we have investigated the response of the Kit tyrosine kinase and several potential cytoplasmic targets to stimulation with Steel in mast cells derived from normal and mutant W mice. In normal mast cells, Steel induces Kit to autophosphorylate on tyrosine and bind to phosphatidylinositol 3'-kinase (PI3K) and phospholipase C-gamma 1 but not detectably to Ras GTPase-activating protein. Additionally, we present evidence that Kit tyrosine phosphorylation acts as a switch to promote complex formation with PI3K. In mast cells from mice homozygous for the W42 mutant allele, Kit is not tyrosine phosphorylated and fails to bind PI3K following Steel stimulation. In contrast, in the transformed mast cell line P815, Kit is constitutively phosphorylated and binds to PI3K in the absence of ligand. These results suggest that Kit autophosphorylation and its physical association with a unique subset of cytoplasmic signaling proteins are critical for mammalian development.

Animals↗
PubMed1991

Contiguous patterns of c-kit and steel expression: analysis of mutations at the W and Sl loci.

Mutations in either the dominant white-spotting (W) or Steel (Sl) loci of the mouse lead to coat color, primordial germ cell and hematopoietic defects. Consistent with the cell autonomous and microenvironmental nature of W and Sl mutations, respectively, it has recently been shown that W encodes the c-kit receptor tyrosine kinase while Sl encodes a ligand for this receptor. Previous in situ hybridization analysis has shown that both c-kit and steel are expressed in the embryo in anatomical sites known to be affected by W and Sl mutations and in various tissues in which no corresponding phenotype has been described. To investigate the possible involvement of the Kit transduction pathway in developmental processes, we compared the patterns of expression of c-kit and steel in wild-type embryos and in embryos homozygous for severe (lethal) and mild (viable) alleles at the W and Sl loci. In addition, we analyzed the patterns of expression of both genes in adult wild-type and mutant gonads and brain. Both c-kit and steel are contiguously expressed in a wide variety of anatomical locations in both the developing embryo and in the adult. In adult gonads, steel is expressed in the follicular cells of the ovary and in Sertoli cells of the testis, the layers that immediately surround the c-kit expressing germ cells. In adult brain, the complementary patterns are particularly striking in the olfactory bulb, cerebral cortex, hippocampus region and cerebellum. steel expression in brain is probably restricted to neurons in certain areas, while c-kit is expressed in neurons and in some glial cells. Severe mutations in the W or Sl loci result in dramatic reduction or absence of c-kit positive cells in lineages known to be affected by these mutations. In contrast, these mutations do not affect the number or histological organization of c-kit positive cells in the embryonic peripheral or central nervous systems, nor is the number or organization of c-kit positive cells detectably altered in Wv/Wv or Sld/Sld adult brain. Taken together, these results suggest that the Kit signaling pathway is not obligatory for the viability and/or migration of most c-kit expressing cells either because of functional redundancy with another signaling pathway or because the Kit pathway is involved in post-developmental processes of mature cells.

Animals↗
PubMed1991

The murine W/c-kit and Steel loci and the control of hematopoiesis.

The experiments summarized here indicate that germ-line mutations in either the c-kit receptor tyrosine kinase (W mutants) or its ligand (Sl) lead to profound and pleiotropic developmental defects, including abnormalities within the hematopoietic system. These observations parallel findings in other developmental systems, notably the fruit fly Drosophila, that receptor tyrosine kinases play key roles in the determination of cell fate and the elaboration of developmental programs. Thus, it appears that the processes of hematopoiesis, melanogenesis, and gametogenesis in mammals involve a similar strategy to that used in other species for transmitting and receiving positional cues during development. Finally, it is interesting to note that what began as an attempt to understand the molecular basis of coat color mutations in the mouse has led to the identification of a key cell-signalling pathway in the development of at least three cell lineages in mammals. Further analysis of the W/Sl pathway should provide a more complete understanding of the early events in hematopoiesis, and may also lead to novel therapeutic applications involving the protein product of the Sl locus.

Animals↗
PubMed1991

p53 transgenic mice: accelerated erythroleukemia induction by Friend virus.

Mutations in the p53 tumor-suppressor gene have been implicated in the pathogenesis of a significant proportion of human cancers and in a dominantly inherited familial cancer syndrome (Li-Fraumeni syndrome). Frequent rearrangements and point mutations have also been detected in the p53 gene in the murine erythroleukemias induced by Friend leukemia virus. We have previously reported that transgenic mice overproducing a mutated p53 protein are predisposed to the development of lung carcinomas, bone and soft-tissue sarcomas, as well as lymphoid malignancies. Here we report that p53 transgenic mice infected with the polycythemia-inducing strain of Friend virus (FV-P) progress to the late stage of erythroleukemia more rapidly than do normal mice. In addition, Friend leukemic cell lines derived from p53 transgenic mice overproduce mutant p53 protein and show a high frequency of rearrangement of the ets-related Spi-1 oncogene, as previously reported in Friend cell lines derived from non-transgenic animals. These results suggest that the same genetic changes involved in the evolution of Friend leukemia in normal mice are also required in mice with an inherited predisposition to cancer. The data also indicate that p53 transgenic mice provide an animal model in which to analyse the role that genetic and environmental factors play in influencing cancer predisposition.

Animals↗