Joel Hayflick - Academia.edu (original) (raw)
Papers by Joel Hayflick
NOVEL PI3K DELTA INHIBITORS AND METHODS OF USE THEREOF
Virology, Jan 30, 1985
The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poor... more The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyade...
Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations
Nature, 1983
Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines express... more Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.
The FASEB Journal, 2006
P110␦ phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of ... more P110␦ phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of certain inflammatory cells. Recent findings revealed that the activity of p110␦ also contributes to allergen-IgE-induced mast cell activation and vascular permeability. We investigated the role of p110␦ in allergic airway inflammation and hyperresponsiveness using IC87114, a selective p110␦ inhibitor, in a mouse asthma model. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of ICAM-1 and VCAM-1 expression, and airway hyperresponsiveness. Intratracheal administration of IC87114 significantly (P<0.05) attenuated OVA-induced influx into lungs of total leukocytes, eosinophils, neutrophils, and lymphocytes, as well as levels of IL-4, IL-5, IL-13, and RANTES in a dose-dependent manner. IC87114 also significantly (P<0.05) reduced the serum levels of total IgE and OVA-specific IgE and LTC 4 release into the airspace. Histological studies show that IC87114 inhibited OVAinduced lung tissue eosinophilia, airway mucus production, and inflammation score. In addition, IC87114 significantly (P<0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. Western blot analyses of whole lung tissue lysates shows that IC87114 markedly attenuated the OVA-induced increase in expression of IL-4, IL-5, IL-13, ICAM-1, VCAM-1, RANTES, and eotaxin. Furthermore, IC87114 treatment markedly attenuated OVA-induced serine phosphorylation of Akt, a downstream effector of PI3K signaling. Taken together, our findings implicate that inhibition of p110␦ signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.-Lee, K. S., Lee, H. K., Hayflick, J. S., Lee, Y. C., Puri, K. D. Inhibition of phosphoinositide 3-kinase ␦ attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J.
Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor
Proceedings of the National Academy of Sciences, 1985
... necrosis factor (cDNA sequence/protein homology/macrophage cytotoxin/necrosin) DIANE PENNICA*... more ... necrosis factor (cDNA sequence/protein homology/macrophage cytotoxin/necrosin) DIANE PENNICA*, JOEL S. HAYFLICK*, TIMOTHY S. BRINGMAN*, MICHAEL A. PALLADINOt, AND DAVID V. GOEDDEL* Departments of ...
Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells
Nature, 1984
The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precur... more The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.
Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin
Nature, 1984
Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, ... more Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, a lymphokine that has similar biological properties. Recombinant tumour necrosis factor can be obtained by expression of its complementary DNA in Escherichia coli and induces the haemorrhagic necrosis of transplanted methylcholanthrene-induced sarcomas in syngeneic mice.
Insulin-like growth factor II precursor gene organization in relation to insulin gene family
Nature, 1984
The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF... more The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.
The Rat Gonadotropin-Releasing Hormone: SH Locus: Structure and Hypothalamic Expression
Molecular Endocrinology, 1989
The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three... more The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5&amp;#39;-sequences spliced to a common 3&amp;#39;-region.
Journal of Leukocyte Biology, 2006
Macrophages detecting and migrating toward sites of injury and infection represent one of the fir... more Macrophages detecting and migrating toward sites of injury and infection represent one of the first steps in an immune response. Here we directly image macrophage birth and migration in vivo in transgenic medaka fish. Macrophages are born as frequently dividing, immotile cells with spherical morphology that differentiate into flat, highly motile cells. They retain mitotic activity while spreading over the entire body. Cells follow restricted paths not only in directed migration, but also during patrolling. Along those paths the macrophages rapidly patrol the tissue and respond to wounding and bacterial infection from long distances. Upon injury they increase their speed and migratory persistence. Specifically targeting PI3kinase isoforms efficiently blocks the wounding response and results in a distinct inhibition of cell motility and chemotaxis. Our study provides in situ insights into the properties of immature and migratory macrophages and presents a unique model to further test modulating compounds in vivo. J. Leukoc. Biol. 81: 263-271; 2007.
Human Molecular Genetics, 2004
Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden -Spatz syn... more Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden -Spatz syndrome) is a rare but devastating neurodegenerative disorder, resulting from an inherited defect in coenzyme A biosynthesis. As pathology in the human condition is limited to the central nervous system, specifically the retina and globus pallidus, we have generated a mouse knock-out of the orthologous murine gene (Pank2 ) to enhance our understanding of the mechanisms of disease and to serve as a testing ground for therapies. Over time, the homozygous null mice manifest retinal degeneration, as evidenced by electroretinography, light microscopy and pupillometry response. Specifically, Pank2 mice show progressive photoreceptor decline, with significantly lower scotopic a-and b-wave amplitudes, decreased cell number and disruption of the outer segment and reduced pupillary constriction response when compared with those of wild-type littermates. Additionally, the homozygous male mutants are infertile due to azoospermia, a condition that was not appreciated in the human. Arrest occurs in spermiogenesis, with complete absence of elongated and mature spermatids. In contrast to the human, however, no changes were observed in the basal ganglia by MRI or by histological exam, nor were there signs of dystonia, even after following the mice for one year. Pank2 mice are 20% decreased in weight when compared with their wild-type littermates; however, dysphagia was not apparent. Immunohistochemistry shows staining consistent with localization of Pank2 to the mitochondria in both the retina and the spermatozoa.
In Vitro Deletional Mutagenesis for Bacterial Production of the 20,000-Dalton Form of Human Pituitary Growth Hormone
DNA, 1983
The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pitui... more The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pituitary glands differs from the major form of hGH (22K, 191 amino acids) by the deletion of amino acid residues 32-46. Using oligonucleotide-mediated mutagenesis, the DNA coding for these amino acids was deleted from the gene previously constructed by us (Goeddel et al., 1979) for microbial hGH production. The DNA to be deleted was looped out by the annealing of a synthetic oligodeoxyribonucleotide to the coding strand of the hGH gene contained on recombinant phage M13 mp8 DNA. Resulting heteroduplex structures were stabilized using primer-directed in vitro DNA synthesis in the presence of T4 DNA ligase. On transformation of Escherichia coli, these heteroduplex DNAs yielded phage whose genomes contained either the original or the partially deleted hGH gene, and genotypes were distinguished by in situ plaque hybridization with synthetic oligonucleotide probes. A gene with the correct deletion was used to express the short hGH variant in E. coli.
Isolation of a cDNA Clone Coding for the γ-Subunit of Mouse Nerve Growth Factor Using a High-Stringency Selection Procedure
DNA, 1984
Page 1. DNA Volume 3, Number 5, 1984 Mary Ann Liebert, Inc., Publishers Isolation of a cDNA Clone... more Page 1. DNA Volume 3, Number 5, 1984 Mary Ann Liebert, Inc., Publishers Isolation of a cDNA Clone Coding for the 7-Subunit of Mouse Nerve Growth Factor Using a High-Stringency Selection Procedure AXEL ULLRICH, ALANE ...
Tissue-Specific and Developmentally Regulated Transcription of the Insulin-Like Growth Factor 2 Gene
DNA, 1987
Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expressio... more Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expression of these genes varies both between tissues and within a given tissue during different stages of development. Alternative splicing or possibly transcriptional initiation events generate variant IGF-2 mRNAs that contain different 5&amp;#39;-untranslated leader sequences. These leader exon sequences are shared with non-IGF-2 mRNAs. Certain noncoding IGF-2 gene sequence elements are transcribed extensively and are found in multiple copies elsewhere in the human genome. Furthermore, IGF-2 mRNA levels are particularly high in a variety of human malignancies.
Upregulated function of phosphatidylinositol-3-kinase in genetically hypertensive rats: a moderator of arterial hypercontractility
Clinical and Experimental Pharmacology and Physiology, 2005
1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediatio... more 1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals. 2. Aorta from spontaneously hypertensive rats (SHR; systolic blood pressure = 183 +/- 4 mmHg) and Wistar Kyoto (WKY) rats (115 +/- 2 mmHg) were isolated for the measurement of isometric contractile force. Aorta from SHR displayed small increases (approximately 5% maximum phenylephrine (PE)-induced contraction) in spontaneous tone, whereas aorta from WKY rats displayed none. The non-selective PI3K inhibitor LY294002 (20 micromol/L) and the selective inhibitor of the p110delta catalytic subunit of PI3K IC87114 (20 micromol/L) caused a fall of basal tone in SHR aorta (20 +/- 7 and 24 +/- 6% of the initial PE contraction, respectively), but did not alter tone in arteries from WKY rats. LY294002, but not IC87114, normalized the increased potency of noradrenaline (NA) observed in aorta from SHR (-log EC50 values for NA in the presence of vehicle in WKY rats and SHR 7.5 +/- 0.1 and 7.8 +/- 0.1, respectively (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.05); -log EC(50) values for NA in the presence of LY294002 in WKY rats and SHR 7.0 +/- 0.1 and 7.0 +/- 0.1, respectively). 3. Biochemical expression of the p110 catalytic and p85 regulator subunits of PI3K in western analyses revealed no difference in expression of the regulatory p85alpha or p110alpha protein subunits between WKY rats and SHR; p110gamma was not detected. In contrast, p110delta expression was increased greater than 30% in aorta from SHR compared with WKY rats (827.6 +/- 88.5 vs 576.8 +/- 53.4 arbitrary densitometry units, respectively). Immunohistochemical analyses revealed expression of the p110delta isoform in the smooth muscle of arteries. 4. These data underscore the relevance of an enzyme historically classified as one committed to growth/anti-apoptosis in modifying contractility and supports involvement of PI3K in genetically based hypertension.
LFA-1 Binding Site in ICAM-3 Contains a Conserved Motif and Non-Contiguous Amino Acids
Cell Communication and Adhesion, 1994
The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that ... more The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-3 and VCAM-1 and hence many form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggest an evolutionary relationship between ICAMs and fibronectin interactions with integrins.
Cancer Research, 2004
The phosphatidylinositol 3-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway p... more The phosphatidylinositol 3-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The ␦ isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110␦ specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110␦ is a therapeutic target to enhance radiation-induced tumor control.
Essential role for the p110 isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia
Blood, 2005
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activa... more The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110alpha, p110beta, p110gamma, and p110delta) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110delta isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110delta-selective inhibitor, suppressed both constitutive and Flt-3-stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110delta as a potential therapeutic target in AML.
Nature, 1985
Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hor... more Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.
NOVEL PI3K DELTA INHIBITORS AND METHODS OF USE THEREOF
Virology, Jan 30, 1985
The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poor... more The transforming (onc) genes of retroviruses contain specific sequences, derived from as yet poorly defined, normal cellular genes, termed proto-onc genes. Proto-onc genes must be defined to explain their docility compared to the oncogenicity of the viral derivatives. Here we set out to determine the borders of the chicken proto-fps gene from which the onc genes of avian Fujinami (FSV) and PRC sarcoma viruses (PRCSV) are derived. These onc genes are hybrids of an element from the gag gene of retroviruses (delta gag) linked to a 2.8-kb domain from proto-fps. To identify the 5' border of proto-fps we have sequenced 1.5 kb beyond the 5' border of overlap with viral fps utilizing a proto-fps clone derived previously. A possible promoter was identified that maps 736 nucleotides from this border. The 736 nucleotides contain two possible exons with 121 codons, and short regions of homology with the delta gag termini of FSV and PRCII. A translation stop codon and an adjacent polyade...
Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations
Nature, 1983
Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines express... more Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.
The FASEB Journal, 2006
P110␦ phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of ... more P110␦ phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of certain inflammatory cells. Recent findings revealed that the activity of p110␦ also contributes to allergen-IgE-induced mast cell activation and vascular permeability. We investigated the role of p110␦ in allergic airway inflammation and hyperresponsiveness using IC87114, a selective p110␦ inhibitor, in a mouse asthma model. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of ICAM-1 and VCAM-1 expression, and airway hyperresponsiveness. Intratracheal administration of IC87114 significantly (P<0.05) attenuated OVA-induced influx into lungs of total leukocytes, eosinophils, neutrophils, and lymphocytes, as well as levels of IL-4, IL-5, IL-13, and RANTES in a dose-dependent manner. IC87114 also significantly (P<0.05) reduced the serum levels of total IgE and OVA-specific IgE and LTC 4 release into the airspace. Histological studies show that IC87114 inhibited OVAinduced lung tissue eosinophilia, airway mucus production, and inflammation score. In addition, IC87114 significantly (P<0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. Western blot analyses of whole lung tissue lysates shows that IC87114 markedly attenuated the OVA-induced increase in expression of IL-4, IL-5, IL-13, ICAM-1, VCAM-1, RANTES, and eotaxin. Furthermore, IC87114 treatment markedly attenuated OVA-induced serine phosphorylation of Akt, a downstream effector of PI3K signaling. Taken together, our findings implicate that inhibition of p110␦ signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.-Lee, K. S., Lee, H. K., Hayflick, J. S., Lee, Y. C., Puri, K. D. Inhibition of phosphoinositide 3-kinase ␦ attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model. FASEB J.
Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor
Proceedings of the National Academy of Sciences, 1985
... necrosis factor (cDNA sequence/protein homology/macrophage cytotoxin/necrosin) DIANE PENNICA*... more ... necrosis factor (cDNA sequence/protein homology/macrophage cytotoxin/necrosin) DIANE PENNICA*, JOEL S. HAYFLICK*, TIMOTHY S. BRINGMAN*, MICHAEL A. PALLADINOt, AND DAVID V. GOEDDEL* Departments of ...
Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells
Nature, 1984
The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precur... more The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.
Human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin
Nature, 1984
Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, ... more Human tumour necrosis factor has about 30% homology in its amino acid sequence with lymphotoxin, a lymphokine that has similar biological properties. Recombinant tumour necrosis factor can be obtained by expression of its complementary DNA in Escherichia coli and induces the haemorrhagic necrosis of transplanted methylcholanthrene-induced sarcomas in syngeneic mice.
Insulin-like growth factor II precursor gene organization in relation to insulin gene family
Nature, 1984
The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF... more The insulin gene family, comprised of insulin, relaxin, insulin-like growth factors I and II (IGF-I and IGF-II) and possibly the beta-subunit of 7S nerve growth factor, represents a group of structurally related polypeptides whose biological functions have diverged. The IGFs, or somatomedins, constitute a class of polypeptides that have a key role in pre-adolescent mammalian growth (see ref. 4 for review). IGF-I expression is regulated by growth hormone and mediates postnatal growth, while IGF-II appears to be induced by placental lactogen during prenatal development. The primary structures of both human IGFs have been determined and are closely related. A polypeptide highly homologous to human IGF-II is secreted by the rat liver cell line, BRL-3A. As this polypeptide, termed multiplication stimulating activity (MSA), differs from human IGF-II by only five amino acid residues, MSA probably represents the rat IGF-II protein. Using molecular cloning techniques, we have isolated cDNA and chromosomal genes coding for the MSA and human IGF-II precursors, respectively. Our data, presented here, indicate that both MSA and human IGF-II are synthesized initially as larger precursor molecules. The deduced preprohormones both have molecular weights (MWs) of 20,100 and contain C-terminal propeptides of 89 amino acid residues, which we have named E-peptides. The organization of the IGF-II precursor gene is discussed in relation to that of other insulin gene family members.
The Rat Gonadotropin-Releasing Hormone: SH Locus: Structure and Hypothalamic Expression
Molecular Endocrinology, 1989
The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three... more The rat GnRH gene as expressed in the central nervous system is comprised of four exons and three introns and spans 4.5 kilobases of genomic DNA. Recently it has been shown that the DNA strand opposite that which is transcribed to produce GnRH mRNA is transcribed in heart to produce a set of transcripts, SH RNAs, which share significant exonic sequences with the GnRH gene. The nucleotide sequence of this locus and approximately 3 kilobases on either side has been determined. Northern analysis of hypothalamic RNA probed with GnRH and SH strand specific probes demonstrate that both GnRH and SH RNAs are present within the preoptic hypothalamus. The cap sites for GnRH and SH transcripts have been localized using polymerase chain reaction technology. Results from these experiments indicate that in the preoptic hypothalamus GnRH transcription initiates from three sites. The majority of GnRH transcripts is spliced efficiently and gives rise to the major class of GnRH mRNA. A second spliced population is present in lower abundance, while a third population is not spliced. The SH gene contains at least two distinct promoters, from which two populations of transcripts are derived containing unique 5&amp;#39;-sequences spliced to a common 3&amp;#39;-region.
Journal of Leukocyte Biology, 2006
Macrophages detecting and migrating toward sites of injury and infection represent one of the fir... more Macrophages detecting and migrating toward sites of injury and infection represent one of the first steps in an immune response. Here we directly image macrophage birth and migration in vivo in transgenic medaka fish. Macrophages are born as frequently dividing, immotile cells with spherical morphology that differentiate into flat, highly motile cells. They retain mitotic activity while spreading over the entire body. Cells follow restricted paths not only in directed migration, but also during patrolling. Along those paths the macrophages rapidly patrol the tissue and respond to wounding and bacterial infection from long distances. Upon injury they increase their speed and migratory persistence. Specifically targeting PI3kinase isoforms efficiently blocks the wounding response and results in a distinct inhibition of cell motility and chemotaxis. Our study provides in situ insights into the properties of immature and migratory macrophages and presents a unique model to further test modulating compounds in vivo. J. Leukoc. Biol. 81: 263-271; 2007.
Human Molecular Genetics, 2004
Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden -Spatz syn... more Pantothenate kinase-associated neurodegeneration (PKAN, formerly known as Hallervorden -Spatz syndrome) is a rare but devastating neurodegenerative disorder, resulting from an inherited defect in coenzyme A biosynthesis. As pathology in the human condition is limited to the central nervous system, specifically the retina and globus pallidus, we have generated a mouse knock-out of the orthologous murine gene (Pank2 ) to enhance our understanding of the mechanisms of disease and to serve as a testing ground for therapies. Over time, the homozygous null mice manifest retinal degeneration, as evidenced by electroretinography, light microscopy and pupillometry response. Specifically, Pank2 mice show progressive photoreceptor decline, with significantly lower scotopic a-and b-wave amplitudes, decreased cell number and disruption of the outer segment and reduced pupillary constriction response when compared with those of wild-type littermates. Additionally, the homozygous male mutants are infertile due to azoospermia, a condition that was not appreciated in the human. Arrest occurs in spermiogenesis, with complete absence of elongated and mature spermatids. In contrast to the human, however, no changes were observed in the basal ganglia by MRI or by histological exam, nor were there signs of dystonia, even after following the mice for one year. Pank2 mice are 20% decreased in weight when compared with their wild-type littermates; however, dysphagia was not apparent. Immunohistochemistry shows staining consistent with localization of Pank2 to the mitochondria in both the retina and the spermatozoa.
In Vitro Deletional Mutagenesis for Bacterial Production of the 20,000-Dalton Form of Human Pituitary Growth Hormone
DNA, 1983
The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pitui... more The 20,000-dalton (20K) variant form of human growth hormone (hGH) present in extracts from pituitary glands differs from the major form of hGH (22K, 191 amino acids) by the deletion of amino acid residues 32-46. Using oligonucleotide-mediated mutagenesis, the DNA coding for these amino acids was deleted from the gene previously constructed by us (Goeddel et al., 1979) for microbial hGH production. The DNA to be deleted was looped out by the annealing of a synthetic oligodeoxyribonucleotide to the coding strand of the hGH gene contained on recombinant phage M13 mp8 DNA. Resulting heteroduplex structures were stabilized using primer-directed in vitro DNA synthesis in the presence of T4 DNA ligase. On transformation of Escherichia coli, these heteroduplex DNAs yielded phage whose genomes contained either the original or the partially deleted hGH gene, and genotypes were distinguished by in situ plaque hybridization with synthetic oligonucleotide probes. A gene with the correct deletion was used to express the short hGH variant in E. coli.
Isolation of a cDNA Clone Coding for the γ-Subunit of Mouse Nerve Growth Factor Using a High-Stringency Selection Procedure
DNA, 1984
Page 1. DNA Volume 3, Number 5, 1984 Mary Ann Liebert, Inc., Publishers Isolation of a cDNA Clone... more Page 1. DNA Volume 3, Number 5, 1984 Mary Ann Liebert, Inc., Publishers Isolation of a cDNA Clone Coding for the 7-Subunit of Mouse Nerve Growth Factor Using a High-Stringency Selection Procedure AXEL ULLRICH, ALANE ...
Tissue-Specific and Developmentally Regulated Transcription of the Insulin-Like Growth Factor 2 Gene
DNA, 1987
Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expressio... more Transcription of the rat and human IGF-2 gene loci is unusually complex. The pattern of expression of these genes varies both between tissues and within a given tissue during different stages of development. Alternative splicing or possibly transcriptional initiation events generate variant IGF-2 mRNAs that contain different 5&amp;#39;-untranslated leader sequences. These leader exon sequences are shared with non-IGF-2 mRNAs. Certain noncoding IGF-2 gene sequence elements are transcribed extensively and are found in multiple copies elsewhere in the human genome. Furthermore, IGF-2 mRNA levels are particularly high in a variety of human malignancies.
Upregulated function of phosphatidylinositol-3-kinase in genetically hypertensive rats: a moderator of arterial hypercontractility
Clinical and Experimental Pharmacology and Physiology, 2005
1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediatio... more 1. The growth enzyme phosphatidylinositol 3-kinase (PI3K) was recently implicated in the mediation of arterial spontaneous tone, an event observed in arteries from hypertensive, but not normotensive, subjects that contributes to changes in total peripheral resistance in the hypertensive state. We have shown this occurrence in experimentally induced models of hypertension. However, because the majority of hypertension is genetically based, it is important to demonstrate a similar change in genetically hypertensive animals. 2. Aorta from spontaneously hypertensive rats (SHR; systolic blood pressure = 183 +/- 4 mmHg) and Wistar Kyoto (WKY) rats (115 +/- 2 mmHg) were isolated for the measurement of isometric contractile force. Aorta from SHR displayed small increases (approximately 5% maximum phenylephrine (PE)-induced contraction) in spontaneous tone, whereas aorta from WKY rats displayed none. The non-selective PI3K inhibitor LY294002 (20 micromol/L) and the selective inhibitor of the p110delta catalytic subunit of PI3K IC87114 (20 micromol/L) caused a fall of basal tone in SHR aorta (20 +/- 7 and 24 +/- 6% of the initial PE contraction, respectively), but did not alter tone in arteries from WKY rats. LY294002, but not IC87114, normalized the increased potency of noradrenaline (NA) observed in aorta from SHR (-log EC50 values for NA in the presence of vehicle in WKY rats and SHR 7.5 +/- 0.1 and 7.8 +/- 0.1, respectively (P &amp;amp;amp;amp;amp;amp;amp;lt; 0.05); -log EC(50) values for NA in the presence of LY294002 in WKY rats and SHR 7.0 +/- 0.1 and 7.0 +/- 0.1, respectively). 3. Biochemical expression of the p110 catalytic and p85 regulator subunits of PI3K in western analyses revealed no difference in expression of the regulatory p85alpha or p110alpha protein subunits between WKY rats and SHR; p110gamma was not detected. In contrast, p110delta expression was increased greater than 30% in aorta from SHR compared with WKY rats (827.6 +/- 88.5 vs 576.8 +/- 53.4 arbitrary densitometry units, respectively). Immunohistochemical analyses revealed expression of the p110delta isoform in the smooth muscle of arteries. 4. These data underscore the relevance of an enzyme historically classified as one committed to growth/anti-apoptosis in modifying contractility and supports involvement of PI3K in genetically based hypertension.
LFA-1 Binding Site in ICAM-3 Contains a Conserved Motif and Non-Contiguous Amino Acids
Cell Communication and Adhesion, 1994
The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that ... more The intercellular adhesion molecule-3 (ICAM-3) is a counter receptor for the integrin LFA-1 that supports cell-cell adhesion dependent functions. ICAM-3 is a member of the immunoglobulin superfamily possessing five immunoglobulin-like domains. Here, we characterize the overall shape of ICAM-3 and the amino acid residues involved in binding LFA-1 and monoclonal antibodies (Mab). Electron microscopic observations show that ICAM-3 is predominantly a straight rod of 15 nm in length, suggesting a head to tail arrangement of the immunoglobulin-like domains. Six out of nine ICAM-3 Mab described blocked the interaction with LFA-1 to varying degrees. Domain assignment of blocking Mab epitopes and characterization of LFA-1-dependent cell adhesion to ICAM-3 mutants demonstrate that the amino-terminal domain of ICAM-3 interacts with LFA-1. A conserved amino acid motif including residues E37 and T38 form an integrin binding site (IBS) in ICAM-3. This motif has also been shown to function as an IBS in ICAM-3 and VCAM-1 and hence many form a common site of contact in all CAMs of this type. Other ICAM-3 residues critical to adhesive interactions, such as Q75, conserved in ICAM-1 and ICAM-2, but not VCAM-1, may confer specificity to LFA-1 binding. This residue, Q75, is predicted to locate in a model of ICAM-3 to the same site as RGD in the immunoglobulin-like domain of fibronectin that binds several integrins. This suggest an evolutionary relationship between ICAMs and fibronectin interactions with integrins.
Cancer Research, 2004
The phosphatidylinositol 3-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway p... more The phosphatidylinositol 3-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The ␦ isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110␦ specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110␦ is a therapeutic target to enhance radiation-induced tumor control.
Essential role for the p110 isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia
Blood, 2005
The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activa... more The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway has been shown to be frequently activated in blast cells from patients with acute myeloid leukemia (AML) and to contribute to survival and proliferation of these cells. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110alpha, p110beta, p110gamma, and p110delta) that are responsible for Akt activation. It is not known which PI3K isoform is critical in AML. Here we show that the p110delta isoform of PI3K is consistently expressed at a high level in blast cells from AML, in contrast to the other class I isoforms, the expression of which was very variable among patients. IC87114, a p110delta-selective inhibitor, suppressed both constitutive and Flt-3-stimulated Akt activation in blasts to the same extent as Ly294002, an inhibitor of all PI3K isoforms. Moreover, IC87114 inhibited AML cell proliferation without affecting the proliferation of normal hematopoietic progenitor cells. These observations identify p110delta as a potential therapeutic target in AML.
Nature, 1985
Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hor... more Inhibin, a specific and potent polypeptide inhibitor of the secretion of follicle-stimulating hormone (FSH), of gonadal origin and thus a potential contraceptive, may constitute a missing link in the mechanism controlling the differential secretion of the pituitary gonadotropins. Inhibin-like bioactivity has been reported in various fluids and extracts of testis and in ovarian follicular fluid. Although there have been several attempts to purify inhibin from seminal plasma, purification from follicular fluid has been more successful (refs 14-16; for review see ref. 17). We have previously isolated two forms (A and B) of inhibin from porcine follicular fluid. Each form comprised two dissimilar subunits of relative molecular mass (Mr) 18,000 (18K, referred to here as the alpha-subunit) and 14K (the beta-subunit), crosslinked by one or more disulphide bridge(s). Forms A and B differ in the N-terminal sequence of their 14K subunit. Preliminary structural characterization of porcine and bovine ovarian inhibins shows that they have similar properties. Here, we have used the N-terminal amino-acid sequence data on the subunits of each inhibin to identify cloned complementary DNAs encoding the biosynthetic precursors and report that inhibins are the product of a gene family that also includes transforming growth factor-beta (TGF-beta) and whose structural organization is similar to that of pituitary and placental glycoprotein hormones.