The future as seen from the biological sciences.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
J D Watson
Publications and source records attributed to J D Watson.
Antigenic proteins of Mycobacterium leprae. Complete sequence of the gene for the 18-kDa protein.
Recombinant clones expressing antigenic determinants of the 18-kDa protein antigen from Mycobacterium leprae recognized by the L5 monoclonal antibody were isolated from a lambda gt11 expression library and their nucleotide sequences determined. All clones expressed the M. leprae-specific determinant as part of a large fusion protein with Escherichia coli beta-galactosidase. The deduced amino acid sequence of the coding region indicated that all the lambda gt11 recombinant clones contained an incomplete M. leprae gene sequence representing the carboxy-terminal two-thirds (111 amino acids) of the 18-kDa gene and coding for a peptide of m.w. 12,432. Subsequent isolation and sequencing of a 3.2kb BamHI-PstI DNA fragment from a genomic M. leprae cosmid library permitted the deduction of the complete 148 amino acid sequence with a predicted m.w. of 16,607. A second open reading frame 560 bases downstream from the 18-kDa coding sequence was found to code for a putative protein of 137 amino acids (m.w. = 15,196). Neither this nor the 18-kDa amino acid sequence displayed any significant homologies with any proteins in the GENBANK, EMBL, or NBRF data bases. Crude lysates from recombinant lambda gt11 clones expressing part of the 18-kDa protein have been reported to stimulate the proliferation of some M. leprae-specific helper T cell clones. Thus, it is significant that the complete 18-kDa sequence contains five short peptides predicted to be possible helper T cell antigenic epitopes based on their propensity to form amphipathic helices. Although three of these occur within the 111 amino acid carboxy-terminal peptide expressed by lambda gt11 clones, the most highly amphipathic peptide is found in the amino-terminal region not present in the lambda gt11 recombinants.
Amino Acid Sequence↗
Effect of granulocyte-macrophage colony-stimulating factor and interleukin 3 on the v-src oncogene. Inhibition of tyrosine kinase activity in the absence of changes in gene expression.
Three cloned murine interleukin 3 (IL-3)-dependent cell lines have been converted to interleukin 2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF) growth-dependent states. FD.C/1 32Dcl-23 and GM cells grown and maintained as IL-3-dependent cell lines, and cells grown with GM-CSF have been infected with a murine recombinant retrovirus containing the v-src oncogene, and grown as lymphokine-independent cell lines. There is a significant increase in tyrosine kinase activity in cells which become lymphokine-independent. FD.C/1 and 32Dcl-23 cells maintained as IL-2-dependent cells lines and infected with the same virus did not grow as IL-2-independent cells. The lymphokine-independent cells FD.C/1src, 32Dsrc, and GMsrc all expressed high levels of tyrosine kinase activity, ranging from 5- to 20-fold more than levels measured in virus-infected cell lines maintained as IL-2-dependent cells. The exposure of FD.C/1src and 32Dsrc cells to IL-3, and GMsrc cells to IL-3 or GM-CSF, resulted in significant decreases in tyrosine kinase activity. These changes were rapidly reversed by removal of IL-3 or GM-CSF from these cells. However, the synthesis of v-src-specific RNA was not affected by the presence of IL-3 or GM-CSF in these cell lines. The biochemical pathways activated by IL-3 or GM-CSF inhibit the activity of the tyrosine kinase encoded by the v-src oncogene without altering gene transcription.
Animals↗
Aminobisphosphonate inhibition of interleukin-1-induced bone resorption in mouse calvariae.
Interleukin-1 (IL-1) is probably an important lymphokine mediator of inflammation and bone resorption. IL-1 derived from mononuclear cells, a melanoma cell line (MM96 cells), and recombinant human IL-1 (rHuIL-1 beta) increased in vitro bone resorption, as measured by the release of 45Ca from cultured mouse calvariae. The 50% maximum active resorption was observed with 0.125 ng/ml or approximately 10(-11) M rHuIL-1 beta. The resorptive action of IL-1 was not entirely dependent on prostaglandin mediation, since its effect was evident when prostaglandin synthesis was inhibited in the cultures by indomethacin. IL-1-induced resorption has been shown to be inhibited by 10(-5) M 3-amino-1-hydroxypropylidene-1-1-bisphosphonate (APD). This inhibition was partially reversed by increasing doses of IL-1. In vitro toxicity studies showed that at concentrations of 10(-4) M, APD inhibited the growth of cultured MM96, murine myelomonocytic P388D1, and rat osteosarcoma UMR 106 cells, but not other mast and lymphoid cell lines. These in vitro observations may have relevance to the use of APD in bone and joint diseases in which inflammation and bone resorption are prominent.
Animals↗
Adrenal vein and arterial levels of catecholamines and immunoreactive metenkephalin in canine endotoxin shock and their response to naloxone.
The alterations in plasma levels of immunoreactive metenkephalin (ir-metenkephalin) and catecholamines in adrenal vein and arterial blood in response to endotoxin, as well as the effects of subsequent naloxone administration, have been investigated in a canine model. Animals were anaesthetised with alpha chloralose and allowed to breathe spontaneously. The left lumbar adrenal vein was cannulated and an intermittent choke allowed retrograde sampling of the adrenal effluent. Severe shock was produced by the administration of a large bolus of E. coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour). One hour after induction of shock the circulating volume was expanded using a colloidal gelatin solution. Thirty minutes later one group of five animals received a bolus of naloxone (2 mg/kg) followed by a continuous infusion of (1.5 mg/kg per hour), while a control group of five animals was given an equivalent volume of isotonic saline. The production of endotoxin shock was associated with marked increases in adrenal vein and systemic levels of adrenaline and noradrenaline. Naloxone administration transiently limited the fall in adrenal vein levels of adrenaline and noradrenaline (P less than 0.05) following volume replacement and was associated with a sustained increase in systemic adrenaline levels (P less than 0.05). Changes in mean arterial pressure confirmed a significant haemodynamic response to naloxone (P less than 0.05). Alterations in ir-metenkephalin levels in the adrenal vein closely followed the changes in catecholamines, whereas arterial levels rose progressively and were unaffected by naloxone. We conclude that in canine endotoxin shock the opiate antagonist naloxone can transiently increase catecholamine levels in the adrenal effluent and produce a more sustained rise in systemic adrenaline levels. Moreover, the adrenal medulla is not the only source of circulating ir-metenkephalin.
Adrenal Glands↗
Neuroendocrine and cardiovascular responses to high-dose corticosteroid therapy in canine endotoxin shock.
The neuroendocrine and cardiovascular responses to endotoxin administration and the effects of subsequent high-dose corticosteroid therapy have been investigated in dogs. Shock was induced in anaesthetised animals by a large bolus of E. coli endotoxin (5 mg/kg) followed by a continuous infusion (2 mg/kg per hour). One hour after induction of shock, the circulating volume was expanded using a colloidal gelatin solution. Fifteen minutes later, one group of five animals received a bolus of methylprednisolone sodium succinate 30 mg/kg, while a control group of five animals was given an equivalent volume of isotonic saline. The administration of endotoxin produced reductions in mean arterial pressure, cardiac index and left ventricular dp/dtmax, together with increases in systemic and pulmonary vascular resistances. These haemodynamic changes were associated with increases in arterial plasma levels of adrenaline, noradrenaline, cortisol, immunoreactive beta-endorphin and immunoreactive metenkephalin. Cardiovascular improvement followed volume replacement and was associated with reductions in circulating catecholamines. No significant haemodynamic or neuroendocrine changes were demonstrated in the 2 h following steroid therapy.
Animals↗
The "tadpole flap"--its role in closure of palatal fistulae.
"Tadpole flaps" have been used in a series of 13 cleft palate patients in attempts to close anterior palatal fistulae over a 5-year period. The technique was successful in eight patients whose fistulae remained closed. The failures are discussed in detail.
Adolescent↗
Preliminary report of a new method of cleft palate repair.
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Cleft Palate↗
The use of a 'universal' yeast expression vector to produce an antigenic protein of Mycobacterium leprae.
This report describes the use of a recombinant yeast expression vector to synthesize and secrete the Mycobacterium leprae 18 kDa antigenic protein. The protein is secreted with a short hydrophilic 'flag' octapeptide fused to its amino-terminus. The fusion protein can be purified directly from yeast culture supernatant through an anti-flag antibody affinity column and the flag octapeptide removed using enterokinase. The method provides a simple and rapid means of obtaining recombinant 18 kDa antigen in quantities suitable for immunological studies.
Antigens, Bacterial↗
Expression of functional human interleukin-2 receptors in murine interleukin-3-dependent cells.
A murine recombinant retrovirus containing the cDNA encoding the human p55 interleukin-2 (IL2)-binding protein was used to insert this gene into a murine interleukin-3 (IL3)-dependent cell line, FD.C/1. Virus-infected cells, maintained in medium supplemented with IL3, expressed human p55 on the cell surface and readily adapted to growth using human IL2. In the presence of human IL2, the synthesis of the endogenous murine p55 binding protein was induced in FD.C/1 cells, making it difficult to determine whether the human p55 protein was actively involved in the process of growth signal transduction. A cloned cell line, FD.huIL2R-2, was identified which grew in the presence of human IL2 but which had lost the ability to synthesize murine p55 protein. Growth of this clone was inhibited by the monoclonal antibody 2A3 which specifically blocked binding of human IL2 to the human p55 binding protein. Analysis of restriction enzyme digests of FD.huIL2R-2 cell DNA revealed that a rearrangement of a murine p55 gene had occurred, implying that virus infection had resulted in the integration of retroviral DNA at a site close to or within a murine p55 gene. If IL2 signal transduction involves binding to a surface heterodimeric receptor for IL2, it is argued that FD.huIL2R-2 cells contain an IL2 receptor complex of murine p70 and human p55 IL2-binding proteins. Alternatively, it is possible that integration of human p55 DNA into a site close to a murine p55 gene may lead to a hybrid p55 IL2-binding protein. If FD.huIL2R-2 cells express murine p70 IL2-binding protein as part of the receptor complex, the inability of cells to grow in murine IL2 implies that IL2 binding to p70 protein alone is insufficient for a growth signal in these cells. FD.huIL2R-2 cells grow at rates similar in IL3- or human IL2-dependent states. It is likely therefore that the biochemical pathways that control each of these lymphokine-dependent growth states are very similar.
Animals↗
Intracellular recognition events eliminate self-reactive T cells.
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Animals↗
Plasma levels of neuropeptide tyrosine Y (NPY) are increased in human sepsis but are unchanged during canine endotoxin shock despite raised catecholamine concentrations.
Alterations in levels of adrenal vein and circulating immunoreactive neuropeptide tyrosine Y (NPY), as well as systemic catecholamine concentrations, have been investigated in the dog after endotoxin administration. Eighteen dogs were anaesthetized with alpha-chloralose and allowed to breathe spontaneously. Severe shock was produced by the administration of a large bolus of Escherichia coli endotoxin followed by a continuous infusion. In two further animals the left lumbar adrenal vein was cannulated and an intermittent choke allowed retrograde sampling of the adrenal effluent. The production of hypodynamic shock was associated with marked increases in systemic catecholamine concentrations but there were no consistent changes in adrenal vein or systemic immunoreactive NPY levels. In contrast, circulating concentrations of immunoreactive NPY were markedly raised above the normal range in five patients with septic shock.
Animals↗
Characterization of circulating pro-opiomelanocortin-related peptides in human septic shock.
The nature of circulating pro-opiomelanocortin (POMC)-related peptides was investigated in patients with a diagnosis of septic shock. Also, changes following administration of methylprednisolone were monitored using established chromatographic techniques and three radioimmunoassays directed towards the N-terminal, mid-portion and C-terminal regions of the precursor. Adrenocorticotrophin and beta-endorphin-like peptides were identified in the circulation. By 60 min after a pharmacological dose of methylprednisolone (30 mg/kg) concentrations of these peptides were reduced and continued to fall up to 180 min. beta-Lipotrophin and N-terminal POMC(1-76) were also detected by the beta-endorphin and gamma 3-MSH assays respectively. The concentrations of these peptides were noticeably reduced only after 180 min. The 31,000 Da MSH/ACTH/beta-endorphin (POMC) and the 22,000 Da MSH/ACTH precursors were also present in the circulation in septic shock and their concentrations increased following steroid treatment.
Adrenocorticotropic Hormone↗
Effects of colony-stimulating factors on leukemia progenitor cells and oncogene expression.
Realising the therapeutic potential of colony-stimulating factors (CSFs) depends upon understanding the biological responses elicited by these regulators in target hemopoietic cells, and determining the biochemical nature of signals transduced through their receptors. These signals can lead to growth, differentiation or the activation of an effector function. CSF-dependent cells maintained in culture resemble pre-leukemic cells and releasing cells from a factor-dependent growth state must be a final step in leukemogenesis. The measurement of metabolic changes in cells following ligand-receptor interactions has thus far failed to reveal the biochemical identity of growth signal transducing events. The patterns of growth responses observed in various CSF-dependent cell lines provide some idea of the relationship of these events for different CSF species. A number of IL-3-dependent cell lines can be switched to an IL-2- or a GM-CSF-dependent growth state. This implies the intracellular pathways activated by signal transduction through their different receptors must be related. The expression of the v-src oncogene in IL-3- and GM-CSF-dependent cells leads to CSF-independent growth, whereas in an IL-2-dependent growth state the expression of v-src in these same cells does not lead to a loss of the requirement for IL-2 for growth. It might be argued that signal transduction through IL-3- or GM-CSF-specific receptors involves a protein tyrosine kinase. However, the addition of IL-3 or GM-CSF to cells expressing v-src results in a decrease in tyrosine kinase activity, suggesting that the effect of IL-3- or GM-CSF-specific signal transduction is to inhibit the expression of tyrosine kinase. It is unlikely that G-CSF signal transduction involves a receptor-associated tyrosine kinase. 32Dcl-23 cells respond to G-CSF by cell division and terminal differentiation, but when these cells are transformed to factor-independent growth following v-src infection, they remain responsive to G-CSF but lose the capacity to terminally differentiate. We have investigated the growth and differentiative responses of a range of human myeloid leukemias to G-CSF, IL-3 and GM-CSF. There is heterogeneity in the responses of different leukemic cells to these growth factors.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Contamination of suction drainage systems in vascular surgery.
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Drainage↗
The differential inhibition of hemopoietic growth factor activity by cytotoxins and interferon-gamma.
The effects of recombinant human tumor necrosis factor (TNF), lymphotoxin (LT), and interferon-gamma (IFN-gamma) on the growth of human hemopoietic progenitor cells in clonal culture have been examined. Colony growth was induced by using granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage colony-stimulating factor (GM-CSF). A suppressive effect of TNF, LT, and IFN-gamma on the development of granulocyte, macrophage, and mixed granulocyte/macrophage colonies was shown. Suppression of colonies formed after stimulation with G-CSF was greater than that observed after stimulation with GM-CSF. In the presence of a monoclonal antibody to TNF, or polyclonal antibodies to either LT or IFN-gamma, the inhibitory effect of the molecule to which the antibody was directed was abrogated. These findings suggest that progenitor cells responsive to G-CSF or GM-CSF have different sensitivities to the effects of TNF, LT, and IFN-gamma. Defining the interactions of growth factors and inhibitors should increase understanding of mechanisms underlying diseases associated with suppression of normal hemopoiesis, and in predicting the effects in vivo of these bioregulatory molecules in clinical medicine.
Cells, Cultured↗
The induction of lymphokine synthesis and cell growth in IL 3-dependent cell lines using antigen-antibody complexes.
Several IL 3-dependent murine bone marrow-derived cell lines can be stimulated to grow with antigen-antibody (Ag.Ab) complexes. The Ag.Ab complexes induced lymphokine gene expression and the synthesis of IL 2, GM-CSF, IL 3, and BSF-1 (IL 4). The lymphokines produced by these IL 3-dependent cells appeared to stimulate their own growth, as both IL 3 and BSF-1 (IL 4) stimulated the growth of IL 3-dependent cells. Ag.Ab complexes also stimulate the growth of primary cultures of bone marrow cells that have been previously activated with IL 3. Normal bone marrow, IL 2-, and GM-CSF-dependent bone marrow cell lines could bind Ag.Ab complexes, but binding did not result in the induction of lymphokine synthesis or cell growth. Hyperimmune serum from mice also stimulated lymphokine synthesis and cell growth in IL 3-dependent cells, and the stimulatory activity was removed by treatment with Staphylococcus aureus protein A, suggesting the presence of Ag.Ab complexes.
Antigen-Antibody Complex↗
Effect of infection with murine recombinant retroviruses containing the v-src oncogene on interleukin 2- and interleukin 3-dependent growth states.
The cloned murine interleukin 3 (IL 3)-dependent cell lines FD.C/1, 32Dc1-23, and KP3 can each be switched to interleukin 2 (IL 2)-dependent growth states. Replication-defective retroviral vectors have been used to introduce the v-src oncogene into each of these cell lines maintained in either an IL 3- or an IL 2-dependent growth state. These cell lines maintained in an IL 3-dependent growth state were converted to lymphokine-independent growth after infection with v-src. These same cells maintained in an IL 2-dependent growth state and infected with v-src maintained strict lymphokine dependence for growth. Another cloned murine IL 3-dependent cell line, GM, can be switched to a granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent growth state. GM cells maintained as IL 3- or GM-CSF-dependent cells readily converted to a lymphokine-independent growth state when infected with v-src. These experiments indicate that either there exist differences in the biochemical mechanisms of signal transduction through the IL 3- and IL 2-specific receptors, or developmental processes associated with the switching of cells to an IL 2-dependent growth state influence expression of the v-src gene product. These cell lines offer new ways not only for analyzing biochemical pathways that regulate cell growth, but also for analyzing the control of oncogene expression.
Animals↗