The interleukin 1 receptor.
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Binding Sites↗
Biomedical subjects
B D Clark
Publications and source records attributed to B D Clark.
Studies on IL-1 receptors on D10S T-helper cells: demonstration of two molecularly and antigenically distinct IL-1 binding proteins.
Receptor binding studies were performed on the interleukin-1 (IL-1) sensitive T-helper cell line D10S, a stable line which proliferates to subfemtomolar concentrations of IL-1 in the absence of mitogens. IL-1 binds in a specific and saturable manner and Scatchard analysis at 4 degrees C reveals one class of binding affinity. On D10S cells, the Kd for IL-1 is 227 pM +/- 80, with 11,000 (range 3,300 to 23,800) sites per cell. EL4.6.1 cells, which are less sensitive to IL-1, bind with a single class of high affinity sites (55 pM; 4,000 sites). D10S cells incubated 18 h with IL-1 display reduced IL-1 receptor (IL-1R) numbers and affinities, consistent with reduced (75%, p less than 0.005) proliferation to subsequent IL-1; preincubation with IL-4 increases the number of IL-1R which is associated with increased (200%, p less than 0.001) proliferation to IL-1. The molecular mass of the major (80 kD) IL-1R binding [125I]IL-1 alpha on D10S cells was consistently observed at 73 kD as compared to the 80 kD molecule on the EL4 cells. On the other hand, crosslinking studies with [125I]IL-1 beta on D10S cells revealed a novel 46 kD band on gradient SDS-PAGE corresponding to a binding protein of 29 to 30 kD, which is antigenically distinct from the 80 kD IL-1R. Crosslinking of D10S or EL4 cells at 4 degrees C in the presence of phytohemagglutinin (PHA) and labeled IL-1 enhanced the appearance of the 30 kD IL-1 binding protein. The findings are consistent with a two-chain model for the IL-1R, although Scatchard analysis did not consistently indicate two classes of affinities. IL-1 binding to the 80 kD protein may form a heteroduplex with the 30 kD IL-1R which could account for the presence of the 120 to 130 kD IL-1 crosslinked proteins observed by several investigators.
Animals↗
Human pro-IL-1 beta gene expression in monocytic cells is regulated by two distinct pathways.
We have previously reported that the pro-IL-1 beta gene is transiently expressed in THP-1 human monocytic leukemia cells after stimulation with bacterial LPS. Herein we show differential pro-IL-1 beta gene transcription in the same cell type using two distinct stimuli. This transcriptional difference is also reflected at the level of intracellular IL-1 beta protein production. In contrast to LPS, a phorbol ester (PMA) induces a stable, non-transient population of mRNA. Furthermore, each induction pathway is operational under conditions where the other is inhibited, suggesting functional independence. Evidence is presented for post-transcriptional regulation of the two responses in THP-1 cells at the level of mRNA stability. We also demonstrate a similar dualistic response in primary monocyte-derived human macrophages as well as the human myelocytic cell line HL-60.
Cell Line↗
The Nucleic Acid Blot Analyzer II. ANALYZE, an image analysis software package for molecular biology.
The Nucleic Acid Blot Analyzer, a new instrument providing high-speed imaging of 32P labeled nucleic acids, captures, stores and presents images in digital form, thus lending itself to rapid data handling and analysis as well as replacing conventional X-ray film autoradiography for many applications. A software package called ANALYZE has been specifically designed for the instrument in order to provide automatic or semi-automatic analysis for molecular biological techniques. The software includes image display manipulation, quantitative and positional analysis, as well as file maintenance utilities. The specific application of the software/hardware to various techniques is presented.
Molecular Biology↗
Transcriptional regulation of the human prointerleukin 1 beta gene.
Interleukin 1 (IL 1) is a protein produced by monocytes in response to certain antigens which produces a wide variety of cellular responses in various tissues. We have studied the regulation of the human proIL-1 beta gene in THP-1 human monocytic leukemia cells. Lipopolysaccharide (LPS) induction of this gene results in an immediate and transient increase of message that rapidly falls to a low, but constant, level within 6 hr. This decrease results from a specific repression of transcription by 2 hr after stimulation. Cycloheximide inhibition of new protein synthesis causes a superinduction of IL 1 message, but does not alter the initial kinetics of message production. This presumably delays the synthesis of a labile transcriptional repressor protein and implies that the proIL-1 beta gene is under the control of both a transcriptional activator and a newly synthesized transcriptional repressor. The transient increase in mRNA production and the sustained low-level synthesis beyond the initial transient response suggest that the IL 1 protein itself may act intracellularly in a manner analogous to that described for several proto-oncogenes and cellular competence factors.
Cell Line↗
Altered expression of a heat shock protein in the mammalian nervous system in the presence of agents which affect microtubule stability.
In vitro incubation of the isolated rabbit retina at elevated temperature results in the synthesis of a heat shock protein of M.W. 74,000 (hsp74). Recently we have demonstrated that this protein is associated with preparations of purified retinal microtubules and intermediate filaments. In order to examine the possibility that hsp74 synthesis is related to cytoskeletal stability, the effects of agents known to specifically affect microtubules were examined using an in vitro retinal system. Taxol, an antimitotic agent which stabilizes microtubules, was found to reduce the level of hsp74 synthesized in response to elevated temperature. Colchicine, a potent microtubule de-stabilizing agent, did not induce hsp74 synthesis in the absence of elevated temperature, however, under heat shock conditions, hsp74 synthesis was elevated in the presence of colchicine. Kinetics of microtubule assembly were similar in preparations isolated from cerebral hemispheres of control and hyperthermic animals however, microtubules from the latter were altered in appearance and exhibited a higher degree of crosslinking.
Alkaloids↗
Serologic and biochemical analysis of HLA B15 and B5 complexes.
Charge heterogeneity of HLA-B15 and HLA-B5 complexes was analyzed by one-dimensional isoelectric focusing (1D-IEF). Frozen peripheral blood lymphocytes were metabolically labeled with 35S methionine. The class I antigens were immunoprecipitated with monoclonal antibody 4E, which detects a determinant shared by HLA-B locus and Aw19-complex antigens. The desialated 1D-IEF banding patterns were correlated to microcytotoxicity data of a panel of donors from a variety of racial groups. Serologic analysis indicated the presence of specific variants: Te76, Te78, and Te79. 1D-IEF analysis clearly showed polymorphism in the B15 and B5 complexes. The Bw62 associated variant Te79 exhibited bands distinct from Bw62. One Bw62 typed donor produced a band that was different from other Bw62 typed cells. A migration pattern difference was discovered between blacks and Caucasians that were typed Bw57. Investigated antigens included HLA-B35, w46, 51, w52, w53, w57, w58, w62, w63, w70, Te76, Te78, and Te79.
Antibodies, Monoclonal↗
Genomic sequence for human prointerleukin 1 beta: possible evolution from a reverse transcribed prointerleukin 1 alpha gene.
We have isolated the human prointerleukin 1 (proIL-1) beta gene from leukocyte and fetal liver libraries. The nucleotide sequence and its gene organization reveals that the proIL-1 beta gene is composed of seven exons with a primary transcription product length of 7,008 nucleotides. The exon sequence agrees well with that of the human proIL-1 beta cDNA. Features of interest within the transcriptional unit include positioned TATA, CAT, and poly-adenylation signals for gene regulation, as well as the signatures of gene duplication via retrotransposition in the form of flanking direct repeats and a genomic poly A tail. The genomic organization of the proIL-1 beta gene with respect to the number and position of exon boundaries is strikingly similar to that of the recently reported human proIL-1 alpha gene. Therefore, we hypothesize that the proIL-1 beta may have arisen by a reverse transcriptase mediated duplication of the related alpha gene.
Base Sequence↗
A retinal heat shock protein is associated with elements of the cytoskeleton and binds to calmodulin.
Elevation of body temperature to a level similar to that attained during fever induces a disaggregation of polysomes in the mammalian retina and induction of a 74K heat shock protein (hsp74). Induced retinal hsp74 copurifies with twice cycled microtubules and also with purified intermediate filaments, is precipitated by antibodies prepared against purified Tau proteins and binds to calmodulin.
Animals↗
Induction of a heat shock protein in the isolated mammalian retina.
The effects of elevated ambient temperature and addition of the psychotropic drug LSD on protein synthesis in the isolated rabbit retina were investigated. Two dimensional gel electrophoresis followed by fluorography of proteins synthesized in vitro demonstrated that synthesis of a heat shock protein of molecular weight 74,000 (74K) was induced by the elevation of temperature and not by the addition of LSD. The appearance of this heat shock protein was shown to be dependent upon the synthesis of new RNA as shown by the addition of actinomycin-D to the incubation medium. The newly synthesized heat shock protein was associated with both nuclear and cytoplasmic fractions.
Animals↗
Expression of biologically active human interleukin 1 subpeptides by transfected simian COS cells.
"Interleukin 1" (IL-1) is a term used to describe the family of macrophage-derived proteins that mediate many immune and inflammatory reactions. We have previously described the molecular cloning and sequencing of the cDNA encoding the predominant (neutral) form of human IL-1, which has been designated IL-1 beta. We report here that transfection of simian COS cells with this cDNA clone results in the transcription of IL-1 mRNA and the synthesis of antibody-neutralizable intracellular IL-1 biological activity. In addition, selective deletion of regions of the IL-1 cDNA judged not to be essential for function, on the basis of conserved sequence homology, resulted in localization of a "core" region responsible for a majority of the biological activity. These results permit mapping the active site of IL-1 to a peptide of 6970 molecular weight located within the carboxyl third (between Met-136 and Gln-197) of the IL-1 precursor.
Animals↗
Restriction endonuclease mapping of three plasmids from Bacillus thuringiensis var. israelensis.
Restriction endonuclease cleavage site maps have been constructed of plasmids pTX14-1, pTX14-2, and pTX14-3 from Bacillus thuringiensis var. israelensis (Bti).
Bacillus thuringiensis↗
Axonal transport of a heat shock protein in the rabbit visual system.
Intraocular injection of [35S]methionine was used to demonstrate the pronounced induction of a 74-kDa heat shock protein in the rabbit retina after a 3 degrees C increase in body temperature was generated by intravenous administration of D-lysergic acid diethylamide. Two-dimensional polyacrylamide gel electrophoresis and fluorography revealed that the induced heat shock protein underwent axonal transport from retinal ganglion cells into the optic nerve and subsequently down the contralateral optic tract to synaptic termini in the visual projection area. Since the heat shock protein took more than 8 days to move down the optic nerve to the superior colliculus, it is transported by slow rather than by fast axonal transport.
Animals↗
Cloning and Expression of an Insecticidal k-73 Type Crystal Protein Gene from Bacillus thuringiensis var. kurstaki into Escherichia coli.
A 75-kilobase plasmid from Bacillus thuringiensis var. kurstaki (HD-244) was associated with the k-73 type insecticidal crystal protein production by mating into B. cereus and subsequent curing of excess plasmids. This plasmid was partially digested with endonuclease R . Sau3A and the fragments were cloned into Escherichia coli (HB101) on vector pBR322. Candidate clones were screened for plasmid vectors which contained the expected insert size (at least 3 kilobases) and then with an enzyme-linked immunosorbent assay, using antisera prepared against electrophoretically purified, solubilized insecticidal crystal protein of 130,000 daltons. Several positive clones were isolated and were analyzed for expression, toxicity, and genetic content by restriction enzyme analysis. Electrophoretic transfer blots of proteins from a candidate E. coli clone, analyzed by enzyme-linked immunosorbent assay, demonstrated a predominant cross-reacting protein of about 140,000 daltons. Ouchterlony analysis also showed a single precipitin band. Extensive bioassays with Manduca sexta larvae revealed that the E. coli clones make toxin with a specific activity (50% lethal dose per microgram of cross-reacting protein) equivalent to that of the parental B. thuringiensis strain or a B. cereus trancipient carrying the toxin-encoding, 75-kilobase plasmid.
Journal Article↗
Doctoral dissertations on hypnosis: 1923-1980.
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Humans↗
Protein synthesis in the mammalian retina following the intravenous administration of LSD.
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Animals↗
Differing susceptibility of young and adult hamster lungs to injury with pancreatic elastase.
A study was undertaken to determine whether there are differences in the initial response of the lungs of young (28 days) and adult (105 days) hamsters to pancreatic elastase treatment. The elastase was given intratracheally (0.1 mg/100 g body weight), and the animals were studied 24 h later. The mean linear intercept of the lungs of young animals increased less than that of adult animals (p less than 0.05). Adult body weights decreased significantly with elastase treatment, whereas those of young elastase-treated animals did not. The lungs of young animals gained proportionately less weight than those of adult animals, suggesting they experienced less hemorrhage and edema. When the volumes, at given transpulmonary pressures, of fluid-filled lungs were expressed as percent predicted, the values for young animals were significantly less than those for adult animals. The most pronounced differences were at low transpulmonary pressures (1 to 3 cm H2O). Volume-pressure hysteresis was not altered by elastase treatment in either the young or adult animals. We conclude that the lungs of adult hamsters are more susceptible to elastase injury than the lungs of young hamsters.
Age Factors↗
Selective effects of LSD and hyperthermia on the synthesis of synaptic proteins and glycoproteins.
Protein synthesis in rabbit brain was inhibited following the intravenous injection of LSD. The incorporation of [35S]methionine into brain microsomal and synaptic fractions was decreased by 35-45% relative to control values. A selective increase was observed, however, in the relative labeling of a protein of molecular weight 75,000. Our previous studies have shown that LSD induces an increase in body temperature (i.e. hyperthermia) in rabbits. When LSD-induced hyperthermia was blocked the general reduction in labeling of microsomal and synaptic proteins was still apparent but the selective increase in relative labeling of the 75,000 dalton protein was not. Induction of hyperthermia by means other than LSD (i.e. elevation of ambient temperature) produced selective increases in the relative labeling of microsomal and synaptic proteins of molecular weight 75,000 and 95,000. These proteins are similar in molecular weight of two of the major 'heat shock' proteins whose synthesis is induced in several cultured cell lines following elevation of ambient temperature. Fractionation of [35S]methionine-labeled synaptic membranes by lectin affinity chromatography and analysis of [3H]fucose labeling patterns indicated that, in contrast to the general reduction in labeling of brain proteins, the synthesis of synaptic glycoproteins was not altered by LSD. The synthesis of glycosylated proteins present in other subcellular fractions was, however, reduced. These results suggest that LSD induced selective changes in the synthesis of brain proteins and that the synthesis of synaptic glycoproteins may be relatively resistant to drug administration.
Animals↗