Effect of a null mutation of the insulin-like growth factor I receptor gene on growth and transformation of mouse embryo fibroblasts.

Fibroblast cell lines, designated R- and W cells, were generated, respectively, from mouse embryos homozygous for a targeted disruption of the Igf1r gene, encoding the type 1 insulin-like growth factor receptor, and from their wild-type littermates. W cells grow normally in serum-free medium supplemented with various combinations of purified growth factors, while pre- and postcrisis R- cells cannot grow, as they are arrested before entering the S phase. R- cells are able to grow in 10% serum, albeit more slowly than W cells, and with all phases of the cell cycle being elongated.

Molecular model of human beta-cell glucokinase built by analogy to the crystal structure of yeast hexokinase B.

Recent studies have shown that mutations in human beta-cell glucokinase that impair the activity of this key regulatory enzyme of glycolysis can cause early-onset non-insulin-dependent diabetes mellitus (NIDDM). The amino acid sequence of human glucokinase has 31% identity with yeast hexokinase, a related enzyme for which the crystal structure has been determined. This homology has allowed us to model the three-dimensional structure of human glucokinase by analogy to the crystal structure of yeast hexokinase B.

Nucleophosmin (NPM) gene rearrangements in Ki-1-positive lymphomas.

The (2;5)(p23;q35) translocation which results in the fusion of the NPM (nucleophosmin) gene on chromosome 5q35 with the novel ALK (anaplastic lymphoma kinase) gene on chromosome 2p23 [S.W. Morris et al.

Structure of the human gene encoding the beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied forms of the beta 2-adrenergic receptor and related G protein-coupled receptors. beta ARK is one of the best characterized members of a growing family of G protein-coupled receptor kinases. In this article we report the isolation and structural organization of the human beta ARK gene.

Suppression of Philadelphia1 leukemia cell growth in mice by BCR-ABL antisense oligodeoxynucleotide.

When injected into SCID mice, the Philadelphia chromosome-positive chronic myeloid leukemia-blast crisis cell line BV173 induces a disease process closely resembling that seen in leukemia patients. At 1 and 3 weeks after injection of 10(6) BV173 cells, CD10+ cells were detected in the bone marrow of the mice, leukemic colonies grew from bone marrow and spleen cell suspensions, and BCR-ABL transcripts were detectable in bone marrow, spleen, peripheral blood, liver, and lungs. Systemic treatment of the leukemic mice with a 26-mer BCR-ABL antisense oligodeoxynucleotide (1 mg/day for 9 days) induced disappearance of CD10+ and clonogenic leukemic cells and a marked decrease in BCR-ABL mRNA in mouse tissues.

A beta-adrenergic receptor kinase dominant negative mutant attenuates desensitization of the beta 2-adrenergic receptor.

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the activated form of the beta 2-adrenergic receptor (beta 2AR) and related G protein-coupled receptors. To further elucidate the role of beta ARK in receptor desensitization, we generated a beta ARK dominant negative mutant by converting an invariant lysine residue in the protein kinase catalytic domain to an arginine. Expressed and purified beta ARK-K220R was able to inhibit wild type beta ARK phosphorylation of the beta 2AR in vitro.

Sequence analysis of the breakpoint cluster region in the ALL-1 gene involved in acute leukemia.

DNA rearrangements caused by chromosome translocations between band 11q23 and various chromosomes can be detected by a single probe, B859, an 859-base pair complementary DNA fragment derived from the human ALL-1 gene. To try to understand why band 11q23 becomes a frequent target of the translocations, we have sequenced the entire breakpoint cluster region, a 8342-base pair BamHI genomic fragment delineated by B859. We found eight Alu repeats located within this region in the same orientation as the ALL-1 gene.

Quantitative trait loci mapping of three loci controlling morphine preference using inbred mouse strains.

Quantitative trait loci mapping was used to identify the chromosomal location of genes which contribute to oral morphine preference (in a two-bottle choice paradigm) of C57BL/6J mice, compared to DBA/2J mice. An F2 intercross of these two strains (606 mice) was phenotyped for morphine preference and those mice demonstrating extreme values for morphine consumption (the highest and lowest 7.7%) were genotyped for 157 murine microsatellite polymorphisms.

Platelet-derived growth factor-induced expression of messenger RNA for the proliferating cell nuclear antigen requires a functional receptor for the insulin-like growth factor I.

Stimulation of quiescent fibroblasts or fibroblast-like cells with growth factors causes a sharp increase in the mRNA levels of several DNA synthesis genes. With most of these genes, the increase in mRNA levels requires at least 2 growth factors [usually platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I)], but the mRNA of the proliferating cell nuclear antigen (PCNA) gene is induced by PDGF only. Since PDGF is known to induce also expression of IGF-I and its receptor, we inquired as to whether the PCNA mRNA induction by PDGF depended on a functional IGF autocrine loop.

Rhenium-labeled somatostatin analog RC-160. 1H NMR and computer modeling conformational analysis.

The solution conformations of RC-160, cyclic D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Val6-Cys7+ ++-Trp8-NH2, an analog of the tumor antiproliferatory neuropeptide somatostatin, and RC-160 labeled with rhenium (Re-RC-160), have been determined by using two-dimensional 1H NMR spectroscopy (600 MHz) and restrained molecular dynamics simulations. Re-RC-160 yields the same average solution conformation as does the apo form with an antiparallel beta-sheet fold and a type II' beta-turn centered at D-Trp4-Lys5. These results indicate that the spatial topography of the side chains essential for somatostatin receptor binding is maintained in Re-RC-160.

The human homologue of the retroviral oncogene qin maps to chromosome 14q13.

Chromosomal mapping of the human QIN gene (renamed FKH2 by the Human Genome Organization Nomenclature Committee) was initially accomplished by correlation of the presence of the QIN locus with specific chromosome regions in a rodent-human hybrid panel. This analysis revealed that the human QIN gene maps to chromosome region 14q11.2-->14q32, between the TCR and IGH loci.

A rationally designed CD4 analogue inhibits experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an acute inflammatory autoimmune disease of the central nervous system that can be elicited in rodents and is the major animal model for the study of multiple sclerosis (MS). The pathogenesis of both EAE and MS directly involves the CD4+ helper T-cell subset. Anti-CD4 monoclonal antibodies inhibit the development of EAE in rodents, and are currently being used in human clinical trials for MS.

Down-regulation of bcl-2 by p53 in breast cancer cells.

bcl-2 and p53 gene products have been both linked to programmed cell death pathways. We have analyzed several human breast cancer cell lines for the expression of bcl-2 and p53. We found an inverse correlation between the expression of the two proteins.

Multiple loci on human chromosome 11 control tumorigenicity of BK virus transformed cells.

BK virus (BKV) is a human papovavirus that readily transforms rodent cells, but not human cells, to a neoplastic phenotype, suggesting that tumor-suppressor functions expressed in human cells control BKV oncogenicity. Transfer of a normal human chromosome 11 to BKV-transformed mouse cells suppresses the malignant phenotype. In this report we map the regions of chromosome 11 involved in tumor suppression.

Phospholipid-stimulated autophosphorylation activates the G protein-coupled receptor kinase GRK5.

G protein-coupled receptor kinases (GRKs) play an important role in mediating agonist-specific desensitization of numerous G protein-coupled receptors. GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction.

DNA strand exchange in the absence of homologous pairing.

The strand exchange reaction that is widely used for in vitro studies on recombination of DNA molecules is generally presumed to result from a preceding homologous pairing step. With the use of a single-stranded circular DNA and a short homologous linear duplex fragment as substrates in a model strand exchange reaction, it was found that modest concentrations of polyethylene glycol or salt promote formation of heteroduplex molecules and strand exchange. When the duplex fragment was partially resected with an exonuclease exposing a short single-stranded stretch on the ends, the reaction was promoted by the addition of commercial bovine serum albumin.

Platelet-derived growth factor increases the activity of the promoter of the insulin-like growth factor-1 (IGF-1) receptor gene.

Stimulation by platelet-derived growth factor (PDGF) is known to increase the number of IGF-I binding sites in cells in culture. We show here that PDGF also increases the levels of IGF-1 receptor mRNA. Using cell lines stably transfected with an expression plasmid in which the reporter luciferase gene is under the control of the rat IGF-1 receptor gene promoter, we find that PDGF increases the activity of this promoter.

Tumor and growth suppression of breast cancer cells by chromosome 17-associated functions.

Losses of functions from chromosome 17 are the most frequent genetic abnormalities in human breast cancer. To assess the biological role of chromosome 17 in the development of breast cancer, we transferred a normal human chromosome 17 to two breast cancer cell lines. No viable clone maintaining an intact chromosome was obtained in either MDA-MB-231 or MCF-7.

Visual arrestin binding to rhodopsin. Intramolecular interaction between the basic N terminus and acidic C terminus of arrestin may regulate binding selectivity.

Visual arrestin plays an important role in quenching phototransduction via its ability to preferentially bind to phosphorylated light-activated rhodopsin (P-Rh*). Recently we proposed a mechanism for the binding of visual arrestin to P-Rh* that helps to explain the nature of the conformational changes in arrestin observed upon binding. This mechanism involves a multisite interaction between arrestin and P-Rh* and implies an interaction between the C-terminal and N-terminal domains of arrestin.

Correlation between E2F-1 requirement in the S phase and E2F-1 transactivation of cell cycle-related genes in human cells.

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis.

Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ.

The Fourier-Green's function and the rapid evaluation of molecular potentials.

Two tasks must be accomplished when calculating the binding modalities and binding energies of two molecules in solution: the calculation of the interaction energy and the calculation of the effects of solvation. It is the competition between the energy of binding and the energy of remaining solvation which determines the binding properties. It is necessary to calculate (or at least approximate in some manner) the partition function in order to make a theoretical estimate of these effects.

Suppression of tumorigenicity of breast cancer cells by microcell-mediated chromosome transfer: studies on chromosomes 6 and 11.

Development of breast cancer has been associated with deletions at multiple chromosomal regions, including 6q, 11p, and 11q. In this study we analyzed the effects of the introduction of chromosomes 6 and 11 on the cell phenotype of the breast cancer cell lines MDA-MB-231 and MCF-7. Chromosome 6 induced alterations of in vitro growth properties and suppressed tumorigenicity of MDA-MB-231 cells.

Transcription activates RecA-promoted homologous pairing of nucleosomal DNA.

RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome.