Showing papers by "Rockefeller University published in 1985"
TL;DR: The data suggest that cachectin/TNF is one of the principal mediators of the lethal effect of endotoxin, and this effect was dose-dependent and was most effective when the antiserum was administered prior to the injection of the endotoxin.
Abstract: A highly specific polyclonal rabbit antiserum directed against murine cachectin/tumor necrosis factor (TNF) was prepared. When BALB/c mice were passively immunized with the antiserum or with purified immune globulin, they were protected against the lethal effect of the endotoxin lipopolysaccharide produced by Escherichia coli. The prophylactic effect was dose-dependent and was most effective when the antiserum was administered prior to the injection of the endotoxin. Antiserum to cachectin/TNF did not mitigate the febrile response of endotoxin-treated animals, and very high doses of endotoxin could overcome the protective effect. The median lethal dose of endotoxin in mice pretreated with 50 microliters of the specific antiserum was approximately 2.5 times greater the median lethal dose for controls given nonimmune serum. The data suggest that cachectin/TNF is one of the principal mediators of the lethal effect of endotoxin.
2,320 citations
TL;DR: A method is described which combines the use of a visual morphological cytotoxicity assay with a quantitative neutral red (NR) spectrophotometric test, for the assessment of the effect of toxic agents on 3T3 cells in culture.
1,805 citations
TL;DR: The effects of 5′ deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, includ ing different plant tissues, are analysed to allow a more direct assessment of deletion effects.
Abstract: Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.
1,674 citations
TL;DR: It is suggested that the ‘cachectin’ and ‘TNF’ activities of murine macrophage conditioned medium are attributable to a single protein, which modulates the metabolic activities of normal as well as neoplastic cells through interaction with specific high-affinity receptors.
Abstract: In mammals, several well-defined metabolic changes occur during infection, many of which are attributable to products of the reticuloendothelial system. Among these changes, a hypertriglyceridaemic state is frequently evident, resulting from defective triglyceride clearance, caused by systemic suppression of the enzyme lipoprotein lipase (LPL). We have found previously that macrophages secrete the hormone cachectin, which specifically suppresses LPL activity in cultured adipocytes (3T3-L1 cells). When originally purified from RAW 264.7 (mouse macrophage) cells, cachectin was shown to have a pI of 4.7, a subunit size of relative molecular mass (Mr) 17,000 and to form non-covalent multimers. A receptor for cachectin was identified on non-tumorigenic cultured cells and on normal mouse liver membranes. A new high-yield purification technique has enabled us to determine further details of the structure of mouse cachectin. We now report that a high degree of homology exists between the N-terminal sequence of mouse cachectin and the N-terminal sequence recently determined for human tumour necrosis factor (TNF). Purified cachectin also possesses potent TNF activity in vitro. These findings suggest that the 'cachectin' and 'TNF' activities of murine macrophage conditioned medium are attributable to a single protein, which modulates the metabolic activities of normal as well as neoplastic cells through interaction with specific high-affinity receptors.
1,167 citations
TL;DR: Dissociation rate measurements indicate a cooperative interaction between USF and TFIID when simultaneously bound to the promoter DNA.
1,043 citations
TL;DR: The molecular cloning and characterization of a gene regulated by rIFN-γ in U937 cells as well as in human mononuclear cells, fibroblasts and endothelial cells is reported here and may be a member of a family of proteins involved in the inflammatory process.
Abstract: Interferons are a family of proteins first identified by their ability to induce cellular resistance to infection by many viruses. In addition to the antiviral properties it shares with the α- and β-interferons, γ-interferon (IFN-γ), a lymphokine secreted by activated T cells, activates macrophages, stimulates B cells, increases fibroblast and endothelial cell resistance to many non-viral intracellular parasites and modulates cell-surface proteins central to immune cell regulation1–13. To identify molecules involved in the IFN-γ response and characterize their modulation, we have isolated genes that are induced following recombinant IFN-γ treatment of U937 cells, a histiocytic lymphoma cell line with monocytic characteristics14,15. We report here the molecular cloning and characterization of a gene regulated by rIFN-γ in U937 cells as well as in human mononuclear cells, fibroblasts and endothelial cells. Messenger RNA from this gene is induced within 30 min of rIFN-γ treatment and demonstrates maximal (>30-fold) accumulation within 5 h. Increased transcription is partly responsible for this accumulation. This gene encodes a protein of relative molecular mass (Mr) 12,378 which has significant amino-acid homology to platelet factor-4 and β-thromboglobulin, two chemo-tatic proteins released by platelets on degranulation. This IFN-γ-inducible protein may be a member of a family of proteins involved in the inflammatory process.
910 citations
TL;DR: The purification of cachectin to apparent homogeneity and characterization of its receptor should facilitate further investigations into the role of Cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
Abstract: Previous studies have indicated that endotoxin and other bacterial and protozoal products can stimulate macrophages to produce a factor that can suppress the activity of the enzyme lipoprotein lipase (LPL), in vivo and in vitro. In the present report we describe the purification of this factor, cachectin, to apparent homogeneity from the conditioned medium of endotoxin-stimulated RAW 264.7 cells. The isolated protein has an isoelectric point of 4.7 and a subunit molecular weight of 17,000. Although cachectin's isoelectric point and molecular weight are similar to those described for interleukin 1, pure cachectin has no leukocyte-activating factor (LAF) activity. Cachectin at a concentration of 10(-11) M has the ability to suppress the LPL activity of the 3T3-L1 adipocyte cell line by 80%. Binding studies using radio-labeled cachectin and 3T3-L1 adipocytes and C2 myotubules revealed approximately 10(4) high-affinity receptors per cell on both cell types (Ka, 3 X 10(9]. Cachectin receptors were also present on liver membranes but were absent on erythrocytes and lymphocytes. The isolation of cachectin and characterization of its receptor should facilitate further investigations into the role of cachectin and other macrophage mediators in the metabolic derangements that occur during infection and cachexia.
854 citations
TL;DR: A sensitive quantitative procedure for assaying viable cells in monolayer cultures is described, conveniently carried out within the same culture, and standardized for use with 96-well microtiter plates and automatic reading with a Dynatech spectrophotometric microplate reader.
Abstract: A sensitive quantitative procedure for assaying viable cells in monolayer cultures is described. The two-component test involves (a) microscopic screening for morphological alterations after an experimental protocol for cytotoxicity studies and (b) quantitation of surviving cells by incubation with the supravital dye neutral red, followed by colorimetric analysis of the dye extracted from the lysosomes of the viable cells. Both assays, conveniently carried out within the same culture, have been standardized for use with 96-well microtiter plates and automatic reading with a Dynatech spectrophotometric microplate reader. The test can be adapted for use with conventional spectrophotometers.
846 citations
Journal Article•
TL;DR: Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding, andCachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells.
Abstract: A highly specific radioreceptor assay for cachectin/tumor necrosis factor (TNF) was utilized to measure the time course of lipopolysaccharide (LPS)-induced hormone production in rabbits. Cachectin/TNF bioactivity was monitored in the same serum samples by measuring lipoprotein lipase (LPL) suppression in 3T3-L1 cells. Cachectin/TNF is produced in large quantities by LPS-treated rabbits without priming by bacillus Calmette Guerin, C. parvum, or other agents. Nanomolar concentrations of the hormone are achieved, with peak levels occurring at 2 hr postinjection; the hormone is rapidly cleared thereafter. In separate studies, mice were used to assess the distribution and metabolic fate of cachectin/TNF. Radioiodinated hormone is cleared from the plasma with a half-life of 6 to 7 min. Studies of the tissue distribution of label after injection demonstrate that liver, kidneys, skin, and gastrointestinal tract take up most of the hormone. Electrophoretic analysis of tissues recovered from injected animals suggests that the hormone is very rapidly degraded after binding.
768 citations
TL;DR: It is shown that ischemic injury to neurons in rat brain is also potentiated by exposure to high physiological titers of glucocorticoids and is attenuated by adrenalectomy.
Abstract: Sustained exposure to glucocorticoids, the adrenocortical stress hormones, is toxic to neurons, and such toxicity appears to play a role in neuron loss during aging. Previous work has shown that glucocorticoids compromise the capacity of neurons to survive a variety of metabolic insults. This report extends those observations by showing that ischemic injury to neurons in rat brain is also potentiated by exposure to high physiological titers of glucocorticoids and is attenuated by adrenalectomy. The synergy between ischemic and glucocorticoid brain injury was seen even when glucocorticoid levels were manipulated after the ischemic insult. Pharmacological interventions that diminish the adrenocortical stress response may improve neurological outcome from stroke or cardiac arrest.
680 citations
TL;DR: The cofactor was purified from the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-sperMidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed.
Abstract: Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual low molecular weight cofactor for activity. The cofactor was purified from the insect trypanosomatid Crithidia fasciculata and identified as a novel glutathione-spermidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis and leishmaniasis.
TL;DR: Administration of estradiol to gonadectomized female, but not male rats, is associated with increased activity of choline acetyltransferase in the medial aspect of the horizontal diagonal band nucleus, the frontal cortex, and CA1 of the dorsal hippocampus.
TL;DR: Clones of complementary DNA from genes whose expression is increased during the differentiation of adipocytes were used to study the molecular basis of cachectin's actions and found that when mature adipocyte were exposed to cachectIn, the messenger RNA's of those genes diminished and rapidly approached the levels present before differentiation.
Abstract: Certain infections and malignancies in mammals cause the development of a condition known as cachexia in which the animal continues to lose weight, often while consuming an adequate diet. When macrophages are stimulated with an endotoxin, they produce a factor or factors, termed cachectin, that inhibits the activity of fat-producing (lipogenic) enzymes in cultured adipocytes. This effect may reflect one of the physiological bases for cachexia. In the present study, clones of complementary DNA from genes whose expression is increased during the differentiation of adipocytes were used to study the molecular basis of cachectin's actions. In the presence of cachectin, the expression of the corresponding genes was reversibly and specifically inhibited. Furthermore, when mature adipocytes were exposed to cachectin, the messenger RNA's of those genes diminished and rapidly approached the levels present before differentiation.
TL;DR: Results suggest that hypothalamic receptors sensitive to PYY and NPY may participate in the control of carbohydrate consumption, with little or no effect on protein or fat consumption.
TL;DR: It is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-As n-NANP-Pro, which is a logical target for vaccine development.
Abstract: Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.
TL;DR: Results from these studies suggest that AGE could act in vivo as a specific signal for recognition and degradation of senescent macromolecules and may ultimately give rise to some of the physiologic changes that occur with normal aging.
Abstract: Proteins that have been modified by long-term exposure to glucose accumulate advanced glycosylation end products (AGE) as a function of protein age. In these studies, we have characterized the interaction of AGE-protein with mouse peritoneal macrophages, using AGE-modified bovine serum albumin (AGE-BSA, prepared by incubation with glucose) as a probe. AGE-BSA was specifically bound to cells at 4 degrees C and was taken up and degraded at 37 degrees C; these processes were concentration dependent and saturable. Competition experiments with AGE-BSA, BSA incubated with phosphate-buffered saline rather than glucose, and yeast mannan demonstrated that macrophages specifically recognize AGE on proteins by a receptor that is completely distinct from the mannose/fucose receptor. Scatchard analysis of AGE-BSA binding data indicated that there are approximately 1.06 X 10(5) receptors per macrophage, with an affinity constant of 1.75 X 10(-11) M. Specific binding of AGE-BSA to the macrophage receptor was competitively inhibited by BSA that had been chemically coupled to a synthetic analogue of the specific AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI-BSA). FFI-BSA was also taken up by macrophages in a concentration-dependent, saturable manner. Prior incubation of macrophages with AGE-BSA failed to influence the subsequent uptake and degradation of added AGE-BSA. Thus, the AGE receptor does not appear to be down-regulated by exposure to AGE-proteins. Results from these studies suggest that AGE could act in vivo as a specific signal for recognition and degradation of senescent macromolecules. Incomplete removal of AGE-proteins by macrophages may ultimately give rise to some of the physiologic changes that occur with normal aging.
TL;DR: A monoclonal antibody labels migrating amacrine cells in late embryonic retinas and defines cell-specific gene expression in relation to migration of one subclass of CNS neurons in adult rat retina.
TL;DR: In an ongoing study of endocrine function in wild olive baboons living freely in Kenya, sustained social stress was associated with suppressed testosterone (T) concentrations in males as discussed by the authors, and the testes appeared to be the principal site of this inhibition; DEX did not alter LHRH-induced pituitary secretion of LH, somewhat attenuated LH bioactivity, but caused a complete suppression of LH-induced testicular secretion of T.
Abstract: In an ongoing study of endocrine function in wild olive baboons living freely in Kenya, sustained social stress was associated with suppressed testosterone (T) concentrations in males. In the present report, the acute stressor of rapid capture and immobilization caused profound and rapid suppression of T concentrations in these individuals. Elevation of cortisol concentrations preceded, and was at least partially responsible for, the declining T concentrations, as dexamethasone (DEX) administration produced a similar suppression. DEX inhibited T secretion, but did not alter its clearance. The testes appeared to be the principal site of this inhibition; DEX did not alter LHRH-induced pituitary secretion of LH, somewhat attenuated LH bioactivity, but caused a complete suppression of LH-induced testicular secretion of T. Considerable individual variation occurred in sensitivity to stress-induced suppression of T concentrations. Some individuals had transient elevations of T concentrations during the poststress hour, although concentrations ultimately declined significantly. These males were also least sensitive to DEX inhibition of LH-induced T secretion. These studies demonstrate acute stress-induced suppression of gonadal function in a population of primates living in their natural habitat. Furthermore, they implicate glucocorticoid actions mostly at the testes as possible underlying endocrine mechanisms for such regulation.
TL;DR: The findings that NPY was effective in the hypothalamus, as opposed to sites anterior, posterior, lateral or dorsal to this structure, suggest a hypothalamic site(s) of action for this neuropeptide.
TL;DR: The background to this work, using canaries, and the most recent findings suggest that neuronal replacement in adulthood is a form of brain repair, though it happens in the absence of an external lesion.
Abstract: My colleagues and I are interested in brain events that control learning. Our work, using canaries, has shown that brain cells can be replaced in adulthood. We suspect that neuronal replacement in adulthood is related to some kinds of learning; it is also a form of brain repair, though it happens in the absence of an external lesion. I will present the background to this work and our most recent findings.
TL;DR: The initial interaction of the signal sequence with the membrane as well as subsequent chain conductance occur in a microenvironment that is accessible to aqueous reagents, suggesting that both processes probably require integral membrane proteins.
TL;DR: In this paper, the authors presented a model, the only successful one among several attempts, in which the first two empirical generalizations can be derived as a consequence of the third, and the model assumes that species are ordered in a cascade or hierarchy such that a given species can prey on only those species below it and can be preyed on by those species above it in the hierarchy.
Abstract: Three recently discovered quantitative empirical generalizations describe major features of the structure of community food webs. These generalizations are: (i) a species scaling law: the mean proportions of basal, intermediate and top species remain invariant at approximately 0.19, 0.53, and 0.29, respectively, over the range of variation in the number of species in a web; (ii) a link scaling law: the mean proportions of trophic links in the categories basal-intermediate, basal-top, intermediate-intermediate, and intermediate-top remain invariant at approximately 0.27, 0.08, 0.30 and 0.35, respectively, over the range of variation in the number of species in a web; and (iii) a link-species scaling law: the ratio of mean trophic links to species remains invariant at approximately 1.86, over the range of variation in the number of species in a web. This paper presents a model, the only successful one among several attempts, in which the first two of these empirical generalizations can be derived as a consequence of the third. The model assumes that species are ordered in a cascade or hierarchy such that a given species can prey on only those species below it and can be preyed on by only those species above it in the hierarchy.
01 Jan 1985
TL;DR: If enough relevant facts can be brought to bear, and hypotheses can be conclusively ruled out, then a strong inference of the correct hypothesis will finally be possible and principles can be stated clearly and can be tested.
Abstract: The ultimate aim in the study of the nervous system is the explanation of behavior: the demonstration of how behavioral responses are produced as a function of nerve cell activity. Even for small bits of neural tissue and restricted aspects of behavior, the number of nerve cells involved is so large and their connections are so complex that large numbers of hypotheses can be imagined. As a result, in the history of beavioral studies, a great deal of “neurologizing” has occurred. Broad speculation about the overall “organization of the brain” and the manner in which it controls behavior has been entertained because, in most cases, the number of facts available to rule out hypotheses has been small. Thus, no comprehensive hypothesis really could be proved. It has seemed necessary to pick a situation where it would be fruitful to gather a great number of behavioral, neuroanatomical, and neurophysiological facts and thus to narrow down the allowable hypotheses to a small number. If enough relevant facts can be brought to bear, and hypotheses can be conclusively ruled out, then a strong inference (Platt, 1964) of the correct hypothesis will finally be possible. In turn, principles can be stated clearly and can be tested.
TL;DR: Plasma levels of testosterone were significantly higher in males exposed to experimental territorial intrusion than they were in controls, supporting the hypothesis that behavioral stimuli emanating from an intruding male can act as supplementary information stimulating secretion of testosterone.
TL;DR: The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned and a synthetic 18-amino acid peptide binds to a monoclonal antibody directed to the CS protein and inhibits the interaction of this antibody with the native protein in sporozoite extracts.
Abstract: The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned. The deduced sequence of the protein consists of 373 amino acids with a central region of 19 tandem repeats of the nonapeptide Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly. A synthetic 18-amino acid peptide containing two tandem repeats binds to a monoclonal antibody directed to the CS protein of Plasmodium vivax and inhibits the interaction of this antibody with the native protein in sporozoite extracts. The portions of the CS gene that do not contain repeats are closely related to the corresponding regions of the CS genes of two simian malarias, Plasmodium cynomolgi and Plasmodium knowlesi. In contrast, the homology between the CS genes of Plasmodium vivax and Plasmodium falciparum, another malaria parasite of humans, is very limited.
TL;DR: The hypoglossal motor neurons that innervate the vocal organ of the male zebra finch show a selective, long-latency response to sound, and these neurons may be perceived as members of a set of vocal gestures and thus distinct from other environmental sounds.
Abstract: The hypoglossal motor neurons that innervate the vocal organ (syrinx) of the male zebra finch show a selective, long-latency (50-millisecond) response to sound. This response is eliminated by lesions to forebrain song-control nuclei. Different song syllables elicit a response from different syringeal motor neurons. Conspecific vocalizations may therefore be perceived as members of a set of vocal gestures and thus distinct from other environmental sounds. This hypothesis is an avian parallel to the motor theory of speech perception in humans.
Journal Article•
TL;DR: In this paper, the authors showed that a specific IL 1 neutralizing antiserum can effectively suppress the adipocyte lipoprotein lipase (LPL) activity when applied to 3T3-L1 cells.
Abstract: Recombinant murine interleukin 1 (rIL 1) inhibits 3T3-L1 cell expression of lipoprotein lipase (LPL) activity when present in exceedingly dilute concentration (less than 10(-15) M) The extreme sensitivity of the adipocyte system to rIL 1 far exceeds that of the standard lymphocyte-activating factor assay However, enzyme suppression is incomplete; even at micromolar concentrations, rIL 1 causes only about a 50% reduction in LPL activity By contrast, cachectin (tumor necrosis factor) achieves nearly complete LPL suppression at subnanomolar concentrations Concentrated solutions of rIL 1 are incapable of competing with radiolabeled cachectin for binding sites on 3T3-L1 cells rIL 1-induced LPL suppression is abolished by the addition of a specific IL 1 neutralizing antiserum to the assay system rIL 1 appears capable of influencing adipocyte expression of LPL, but apparently acts through a different mechanism than cachectin/TNF
TL;DR: The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.
Abstract: The transforming protein of Rous sarcoma virus, p60src, was shown to be acylated at its amino terminus with the long-chain fatty acid myristic acid by isolation of a tryptic peptide with the following structure: myristylglycylserylseryllysine. The occurrence of this unusual posttranslational modification in the cyclic adenosine monophosphate-dependent protein kinase and in several transforming protein kinases of mammalian retroviruses suggests that myristylation of the amino terminal glycyl residue may be critical for the function of certain proteins related to cell transformation and growth control.
TL;DR: It appears that corticosterone regulation of its own receptors emerges only by about the time of puberty, and that this permits an increase in receptor levels to occur despite the concurrently increasing levels of circulating cortic testosterone.