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D L Simmons

Publications and source records attributed to D L Simmons.

At least 91 records · Page 5Linked to original sources
PubMed1992

Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes.

The co-ordinated function of effector and accessory cells in the immune system is assisted by adhesion molecules on the cell surface that stabilize interactions between different cell types. Leukocyte function-associated antigen 1 (LFA-1) is expressed on the surface of all white blood cells and is a receptor for intercellular adhesion molecules (ICAM) 1 and 2 (ref. 3) which are members of the immunoglobulin superfamily. The interaction of LFA-1 with ICAMs 1 and 2 provides essential accessory adhesion signals in many immune interactions, including those between T and B lymphocytes and cytotoxic T cells and their targets. In addition, both ICAMs are expressed at low levels on resting vascular endothelium; ICAM-1 is strongly upregulated by cytokine stimulation and plays a key role in the arrest of leukocytes in blood vessels at sites of inflammation and injury. Recent work has indicated that resting leukocytes express a third ligand, ICAM-3, for LFA-1 (refs 11, 12). ICAM-3 is potentially the most important ligand for LFA-1 in the initiation of the immune response because the expression of ICAM-1 on resting leukocytes is low. We report the expression cloning of a complementary DNA, pICAM-3, encoding a protein constitutively expressed on all leukocytes, which binds LFA-1. ICAM-3 is closely related to ICAM-1, consists of five immunoglobulin domains, and binds LFA-1 through its two N-terminal domains.

Amino Acid Sequence↗
PubMed1992

Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24.

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.

Amino Acid Sequence↗
PubMed1992

Hormonal regulation of messenger ribonucleic acid encoding a novel isoform of prostaglandin endoperoxide H synthase in rat preovulatory follicles. Induction in vivo and in vitro.

Recent purification and amino-terminal analysis of the isoform of prostaglandin endoperoxide H synthase (PGS) induced in rat preovulatory follicles by gonadotropins identified it as a novel distinct isoform of PGS (rPGSi) which exhibited a high degree of homology to the deduced sequence of PGS-related cDNAs recently cloned in chicken and mice. To further verify the similarities of these novel gene products and to examine the hormonal regulation of rPGSi mRNA in ovarian cells, three different in vivo and in vitro models were used. Northern blots using a cDNA encoding the mouse homologue of rPGSi detected a 4.4-kilobase transcript which was rapidly but transiently induced in granulosa cells of preovulatory follicles exposed in vivo to an ovulatory dose of human chorionic gonadotropin. The rPGSi mRNA was undetectable at 0 h, peaked 4 h after human chorionic gonadotropin, and had almost disappeared by 6 h. Increases in rPGSi protein (immunoblots) lagged by about 1 h, peaked at 5 h, and remained present at 11 h. PGSi mRNA and protein were also induced in a time- and dose-dependent manner when preovulatory follicles were isolated and incubated with elevated levels of follicle-stimulating hormone (500 ng/ml) or luteinizing hormone (500 ng/ml), or when differentiated granulosa cell cultures were stimulated with follicle-stimulating hormone, luteinizing hormone, or with gonadotropin-releasing hormone (10(-6) M). In both in vitro systems, rPGSi mRNA peaked at 4-5 h. When the same RNA samples were probed with the mouse cDNA encoding the other PGS isoform, no mRNA transcripts (2.8 kilobases) were observed. These results show for the first time that a rapid and transient induction of mRNA encoding a novel PGS enzyme occurs in granulosa cells of preovulatory follicles prior to ovulation and that results in vitro closely mimicked those in vivo and thereby provide models for studying the molecular mechanisms of rPGSi gene expression.

Animals↗
PubMed1992

Cis-acting sequences regulating expression of the human alpha-globin cluster lie within constitutively open chromatin.

Current models suggest that tissue-specific genes are arranged in discrete, independently controlled segments of chromatin referred to as regulatory domains. Transition from a closed to open chromatin structure may be an important step in the regulation of gene expression. To determine whether the human alpha-globin cluster, like the beta-globin cluster, lies within a discrete, erythroid-specific domain, we have examined the long-range genomic organization and chromatin structure around this region. The alpha genes lie adjacent to at least four widely expressed genes. The major alpha-globin regulatory element lies 40 kb away from the cluster within an intron of one of these genes. Therefore, unlike the beta cluster, cis-acting sequences controlling alpha gene expression are dispersed within a region of chromatin that is open in both erythroid and nonerythroid cells. This implies a difference in the hierarchical control of alpha- and beta-globin expression.

Animals↗
PubMed1992

Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells.

CD34 is a 115-kDa transmembrane glycoprotein of unknown function that is expressed on human hematopoietic progenitor cells and the small vessel endothelium of a variety of tissues. We have isolated a CD34 cDNA clone from a KG-1 cell library following three rounds of transient expression in COS cells and enrichment by panning with the anti-CD34 mAb MY10 and BI-3C5. The 5' and 3' ends of the full-length cDNA were subsequently amplified by polymerase chain reaction from KG-1 RNA; the final cDNA clone contained 2615 bp and ended in a poly(A) tail. COS cells transfected with the cDNA clone expressed a surface protein of approximately 110 kDa that was immunoprecipitated by MY10. Southern blot analysis suggested that CD34 is a single copy gene. A 2.7-kb CD34 transcript was observed in the hematopoietic cell lines KG-1, KMT-2, AML-1, RPMI 8402, and MOLT 13 and the endothelial cells BAE and EAhy926, but not in monocytes, resting T cells, or the cell lines Laz 509, HL-60, U937, K562, and HeLa. The cDNA sequence predicts a 40-kDa type I integral membrane protein with nine potential N-linked and numerous potential O-linked glycosylation sites in its extracellular domain. There are two consensus protein kinase C phosphorylation sites and one potential tyrosine kinase phosphorylation site in the cytoplasmic portion of CD34. CD34 has no significant sequence homology to any known protein but has some structural similarities to the heavily glycosylated leukocyte surface molecule CD43.

Amino Acid Sequence↗
PubMed1992

Identification of an early-growth-response gene encoding a novel putative protein kinase.

Early-growth-response genes, also known as immediate-early genes, play important roles in regulating cell proliferation. We have identified a new type of early-growth-response gene product, a 77,811-Da putative serine/threonine kinase, which is highly inducible by serum and phorbol ester. mRNA encoding this putative kinase is markedly elevated within 1 h after treatment with mitogen, and this induction is synergistically increased by cycloheximide. Dexamethasone blocks serum induction of the kinase mRNA, as does transformation by v-Ki-ras. The kinase mRNA was detected in mouse brain, lung, and heart. This new putative kinase, which we term Snk, for serum-inducible kinase, showed similarity in its proposed catalytic domain to many other protein kinases; however, no other kinase showed enough sequence similarity with Snk to suggest the existence of a common function. Hence, Snk represents a new type of protein kinase involved in the early mitogenic response whose activity is transcriptionally and posttranscriptionally regulated.

3T3 Cells↗
PubMed1991

Rel-associated pp40: an inhibitor of the rel family of transcription factors.

The Rel-associated protein pp40 is functionally related to I kappa B, an inhibitor of the transcription factor NF-kappa B. Purified pp40 inhibits the DNA binding activity of the NF-kappa B protein complex (p50:p65 heterodimers), p50:c-Rel heteromers, and c-Rel homodimers. The sequence of the complementary DNA encoding pp40 revealed similarity to the gene encoding MAD-3, a protein with mammalian I kappa B-like activity. Protein sequencing of I kappa B purified from rabbit lung confirmed that MAD-3 encodes a protein similar to I kappa B. The sequence similarity between MAD-3 and pp40 includes a casein kinase II and consensus tyrosine phosphorylation site, as well as five repeats of a sequence found in the human erythrocyte protein ankyrin. These results suggest that rel-related transcription factors, which are capable of cytosolic to nuclear translocation, may be held in the cytosol by interaction with related cytoplasmic anchor molecules.

Amino Acid Sequence↗
PubMed1991

Expression of a mitogen-responsive gene encoding prostaglandin synthase is regulated by mRNA splicing.

Rous sarcoma virus was shown to induce in chicken embryo fibroblasts (CEF) a 4.1-kilobase mRNA (designated CEF-147) encoding a 603-amino acid protein. Analysis of the protein sequence showed that it shared 59% amino acid identity with sheep prostaglandin G/H synthase, the enzyme that catalyzes the rate-limiting steps in the production of prostaglandins. Significant differences, at both the protein and mRNA levels, existed between the src oncogene product-inducible prostaglandin synthase and the protein isolated and cloned from sheep seminal vesicle, suggesting that the src-inducible prostaglandin synthase may be a new form of the enzyme. A distinguishing feature of src-inducible prostaglandin synthase mRNA is its low abundance in nonproliferating chicken embryo fibroblasts and its relatively high abundance in src-transformed cells. Additionally, the majority of the src-inducible prostaglandin synthase RNA present in nonproliferating cells was found to be nonfunctional because of the presence of an unspliced intron that separated the signal peptide from the remainder of the protein. Upon mitogenic stimulation, this intron was removed, resulting in the induction of fully-spliced CEF-147 mRNA.

Amino Acid Sequence↗
PubMed1990

Molecular cloning of CD31, a putative intercellular adhesion molecule closely related to carcinoembryonic antigen.

cDNA clones encoding CD31 have been isolated by transient expression. The sequence of CD31 expressed on human umbilical vein endothelial cells (HUVEC) is identical to that expressed on the monocyte-like cell line HL60. In HUVEC. CD31 is concentrated in regions of cell-cell contacts. CD31 is a member of the Ig superfamily and is most closely related to the carcinoembryonic antigen CEA, consisting of four contiguous C2 domains. The localization of CD31 to regions of cell-cell contacts, and the sequence similarity to CEA, a known intercellular adhesion molecule (ICAM), strongly suggest that CD31 may function as an ICAM, possibly mediating endothelial cell-cell contacts and also promoting interactions between leukocytes and endothelial cells.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of a chicken c-rel cDNA: unlike p59v-rel, p68c-rel is a cytoplasmic protein in chicken embryo fibroblasts.

We isolated and sequenced a 3727 bp clone of the c-rel proto-oncogene from a chicken embryo fibroblast (CEF) cDNA library. Sequence comparison to the retroviral oncogene v-rel showed conclusively that the v-rel protein is truncated at both the amino- and carboxy-termini as compared to the c-rel protein. In vitro transcription and translation of this clone yielded a 68,000 dalton polypeptide that co-migrated on SDS polyacrylamide gels with p68c-rel from avian spleen cells. We inserted this c-rel cDNA clone into an avian retroviral vector (pJD214c-rel), and over-expressed p68c-rel in CEF. Over-expression of p68c-rel did not induce morphological transformation of these cells. Unlike p59v-rel, which is a nuclear protein in CEF, indirect immunofluorescence showed that p68c-rel in JD214c-rel infected CEF is located exclusively in the cytoplasm of these cells, even though the sequence of p68c-rel showed that it contains a nuclear localizing sequence identical to the one previously identified in p59v-rel. Furthermore, the c-rel protein does contain a nuclear localizing sequence which can function in CEF since replacement of the v-rel nuclear localizing sequence with the homologous domain from c-rel resulted in a hybrid rel protein that was located in the nucleus in CEF. Mutant c-rel proteins, deleted of the carboxy-terminal sequences not present in p59v-rel, localized to the nucleus in CEF. Our results show that the carboxy-terminus of p68c-rel inhibits nuclear localization of the protein, and suggest that subcellular location may be a form of regulation of the activity of p68c-rel.

Amino Acid Sequence↗
PubMed1989

Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum.

The primary event in the pathogenesis of severe malaria in Plasmodium falciparum infection is thought to be adherence of trophozoite- and schizont-infected erythrocytes to capillary endothelium, a process called sequestration. Identifying the endothelial molecules used as receptors is an essential step in understanding this disease process. Recent work implicates the membrane glycoprotein CD36 (platelet glycoprotein IV; refs 2-5) and the multi-functional glycoprotein thrombospondin as receptors. Although CD36 has a widespread distribution on microvascular endothelium, it may not be expressed on all capillary beds where sequestration occurs, especially in the brain. The role of thrombospondin in cell adhesion, in vitro or in vivo, is less certain. We have noticed that some parasites bind to human umbilical-vein endothelial cells independently of CD36 or thrombospondin. To screen for alternative receptors, we have developed a novel cell-adhesion assay using transfected COS cells, which confirms that CD36 is a cell-adhesion receptor. In addition, we find that an endothelial-binding line of P. falciparum binds to COS cells transfected with a complementary DNA encoding intercellular adhesion molecule-1. As this molecule is widely distributed on capillaries and is inducible, this finding may be relevant to the pathogenesis of severe malaria.

Antibodies, Monoclonal↗
PubMed1989

CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells.

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.

Antibodies, Monoclonal↗
PubMed1989

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development.

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixed-function oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.

Adrenal Glands↗
PubMed1989

Identification of a phorbol ester-repressible v-src-inducible gene.

Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60v-src-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, we have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) induced mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.

Amino Acid Sequence↗
PubMed1989

Monocyte antigen CD14 is a phospholipid anchored membrane protein.

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.

Amino Acid Sequence↗
PubMed1988

Molecular analyses of gene products associated with the response of cells to mitogenic stimulation.

We have completed the molecular analysis of a ribosomal protein S6 kinase identified in unfertilized Xenopus eggs. Although some of our results support the notion that an antigenically related enzyme is present in animal cells, we are uncertain if such an enzyme accounts for all of the mitogen-stimulated phosphorylation of 40S subunits. Other experiments have led to the identification of mRNAs that are rapidly expressed in mitogen-stimulated cells. The relevance of such genes and their products is in doubt, however, until a function can be demonstrated for each of them. Preliminary experiments with the pCEF-4 gene product described here have failed to show that it acts as a mitogen for cells in culture, for example. Thus, although studies such as these may lead to the identification of novel genes, we may need to search elsewhere for a physiologically significant function.

Amino Acid Sequence↗
PubMed1987

Induction of the hepatic mixed-function oxidase system by synthetic glucocorticoids. Transcriptional and post-transcriptional regulation.

Quantitative evaluation of transcriptional rates in isolated nuclei and intranuclear and cytoplasmic mRNA levels demonstrates that the catatoxic steroids pregnenolone-16 alpha-carbonitrile (PCN) and dexamethasone modulate microsomal drug metabolizing enzymes both positively and negatively at the transcriptional level. Additionally, a strong post-transcriptional influence, believed to be message stabilization, was found to be exerted by dexamethasone. Both NADPH-cytochrome P-450b-homologous mRNA are induced 7- and 12-fold, respectively, presumably through message stabilization. Under these conditions, however, the oxidoreductase content of the cell is only marginally increased while the cytochrome P-450b level is actually decreased by 40% within three days after a single injection of dexamethasone. In contrast, genes coding for cytochromes P-450PCN show a definite increase in transcription following administration of either pre gnenolone-16 alpha-carbonitrile or dexamethasone, whereas transcription of the epoxide hydrolase gene is markedly reduced by administration of these compounds.

Animals↗