The high-level expression of human interleukin-la in Escherichiu coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion-and cation-exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N-and C-terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vifro assays, namely, the mononuclear cell factor (IL-1/MCF) assay and lymphocyte activating factor (IL-1 /LAF) assay. The specific activities determined with the IL-1/MCF and IL-l/ LAF assays, are 2 x lo7 and 4 x lo7 units mg-', respectively.The term interleukin-1 (IL-1) applies to at least two cytokines which play a role in immunity and in the pathophysiology of inflammation [I -31. Preparations of authentic IL-1 tend to be contaminated with other cytokine mediators which can complicate the interpretation of sensitive IL-1 bioassays (see, for example [4]). The availability of IL-1 produced by recombinant DNA technology would eliminate the problem of potential contamination and provide a reliable source of well characterized cytokine.Recently, the coding sequences for human IL-la and have been cloned from peripheral blood monocytes and their expressed products shown to possess IL-1 activities [5, 61. Using published sequence data we have isolated clones of IL-lP cDNA from a library constructed from mRNA expressed in human histiocytic lymphoma U937 cells. Here we report the expression of the mature IL-la protein in Escherichiu coli, a method for its purification, and the physiochemical and biological properties of the purified protein. MATERIALS AND METHODS Bacterial hosts and plusmids
Twenty-two new alkenyldiarylmethanes (ADAMs) were synthesized and evaluated for inhibition of HIV-1 replication. The most potent compound proved to be methyl 3',3"-dichloro-4',4"-dimethoxy-5', 5"-bis(methoxycarbonyl)-6,6-diphenyl-5-hexenoate (ADAM II), which displayed an EC50 of 13 nM for inhibition of the cytopathic effect of HIV-1RF in CEM-SS cells. ADAM II inhibited HIV-1 reverse transcriptase with an IC50 of 0.3 microM but was inactive as an inhibitor of HIV-1 attachment/fusion to cells, protease, integrase, and the nucleocapsid protein. Molecular target-based and cell-based assays revealed that ADAM II acted biologically as a nonnucleoside reverse transcriptase inhibitor (NNRTI). ADAM II inhibited replication of a wide variety of laboratory, clinical, and clade-representative isolates of HIV-1 in T cell lines and cultures of peripheral blood mononuclear cells or monocyte/macrophages. Mutations that conferred resistance to ADAM II clustered at residues 101, 103, 108, 139, 179, 181, and 188, which line the nonnucleoside binding pocket of HIV-1 reverse transcriptase. However, HIV-1 NL4-3 strain expressing a mutation at residue 100 of reverse transcriptase, and an AZT-resistant virus, displayed increased sensitivity to ADAM II. Thus, ADAM II could serve as an adjunct therapy to AZT and NNRTIs that select for L100I resistance mutations.
Interleukin 1 (IL-1) possesses multiple biological activities that may be blocked selectively by different inhibitors. Some known inhibitors block the lymphocyte activating factor (LAF/IL-1) but not the mononuclear cell factor (MCF/IL-1) measured by its capacity to stimulate prostaglandin E2 (PGE2) and colingenase production. The presence of IL-1 in vivo may be difficult to detect due to the presence of inhibitor(s) and the level of the inhibitor(s) may vary depending upon pathological conditions. We have found that urine from three patients with monocytic leukemia (M5) contained high levels of inhibitor(s) of MCF/II-1, whereas urine of normal subjects did not contain significant amounts. Urine from two patients with other blood neoplasic diseases also contained little inhibitory activity. The MCF/IL-1 inhibitor(s), which also acts on human recombinant IL-1 ft, is -25-35 kD, is not retained on concanavalin A-Sepharose column and can be partially destroyed with urea and boiling. At this stage of the purification the fraction containing the MCF/IL-1 inhibitor(s) also inhibits the LAF/IL-1 assay. However, this inhibitor(s) is probably distinct from other inhibitors already described.
The pharmacokinetics of heparin differ markedly from those of its fractions both in man and in experimental animal models. The route of administration determines the relative availability of different molecular species that exert the anti-Xa, anti-IIa, fibrinopeptide A generation inhibiting actions and the release of tissue plasminogen activator-like activity from the endothelial cell lining. The bioavailability of heparin fractions has proved to be much greater than heparin after subcutaneous or intraperitoneal administration. Most of the low molecular weight heparin fractions exhibit sustained antiprotease and antithrombotic actions. The pharmacokinetics of the specific anti-IIa and anti-Xa actions of heparin and its fractions is dependent on the molecular composition of these agents. Even if the fractions are standardized for identical potencies by the in vitro assays, the elimination rate of anti-Xa and anti-IIa actions are significantly different for each fraction. The antithrombotic actions of heparin and its fractions also vary widely in the rabbit stasis thrombosis model. Different fractions show variable antithrombotic actions against defined thrombogenic challenges. Moreover, selection and potency of a thrombogenic agent is of crucial importance in these studies. The primate (Macaca mulatta) model offers a useful preclinical model for the pharmacologic evaluation of the low molecular heparin fractions. Since the coagulation system and heparinizability index of this model approximate a human response, the data may be used to reflect therapeutic and prophylactic responses, as well as to assess toxic effects, such as bleeding.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.