Adriamycin-induced activation of NK activity may initially involve LAF production

C3HeB/FeJ spleen cells (unseparated or passaged over nylon wool columns) were cultured overnight (1-2 X 10(6) cells/microwell) in the presence and absence of resident or ADM-induced PEC and anti-YAC-1 (4h) NK activity was determined. The addition of resident PEC to spleen cells had little effect on NK activity. However, the addition of ADM-elicited PEC (10 mg/kg, IP, day -1 and day -5) to spleen cells prior to culture significantly augmented NK activity. If ADM-induced PEC were treated with carbonyl iron prior to coculture with spleen cells, augmentation of anti-YAC-1 activity was not observed. This suggested that ADM-activated macrophages augmented cultured splenic NK activity. Supernatants from overnight-cultured resident or ADM-induced adherent PEC were then prepared, dialyzed (to remove ADM), and tested for mitogenic activity or cocultured with spleen cells overnight. ADM-induced adherent PEC supernatants stimulated the proliferation of murine thymocytes (both LAF and IL-2 also stimulate) but not cultured CTL (only IL-2 stimulates). ADM-induced adherent PEC supernatants (as well as LAF, IL-2, and IFN) augmented overnight-cultured C3HeB/FeJ splenic NK activity. However, only IL-2 and IFN could augment overnight-cultured athymic BALB/c . nu/nu splenic NK activity. This suggested that ADM-elicited macrophages produce LAF which may act directly on NK cells or, more likely, may induce T cells to produce IL-2, IFN, or both.     

IGG-stimulated and LPS-stimulated monocytes elaborate transforming growth factor type beta (TGF-beta) in active form

Mononuclear cells (MNC) stimulated either with lipopolysaccharide (LPS) or with surface-adsorbed IgG elaborated significant amounts of tumor necrosis factor (TNF) bioactivity, as well as immunoenzymatically detectable TNF-alpha and interleukin-1 beta. (IL1-beta). In contrast, IgG-stimulated cells released little IL1 bioactivity, but released an IL1 inhibitor, as determined by the thymocyte costimulatory assay (LAF assay). This inhibition was not due to an inhibitory effect of cyclooxygenase products, e.g. prostaglandin-E2 in the LAF assay. In contrast, antibodies against transforming growth factor type beta (TGF-beta), which is an important inhibitor of the LAF assay, augmented the LAF activity of supernatants from LPS-stimulated and IgG-stimulated MNC. Anti-TGF-beta-modulated LAF inhibition was enhanced by acid treatment of supernatants from mononuclear cells, but not of those from purified monocytes. Antibody blocking experiments point for the first time to a TGF-beta species other than type 1 as a monocyte-derived TGF-beta activity. Thus, TGF-beta released in active form from monocytes may be the more important antagonist of IL1 than cyclooxygenase-derived mediators. It implies that the LAF assay, in the absence of anti-TGF-beta antibodies, is an inadequate indicator of IL1 activity.     

Constitutive production of lymphocyte activating factors by normal tissues in the adult rat

Lymphocyte activating factors (LAFs), e.g., interleukin-1 (IL-1) and IL-1-like factors, have previously been demonstrated outside the immune system in the skin, thymus epithelium, and the human and rat testis. We have studied the presence of LAFs in normal tissues of the adult rat, utilizing a highly IL-1 sensitive murine thymocyte proliferation assay. We have demonstrated high amounts of LAF activity in the tongue, esophagus, proventricular part of the stomach, and the liver. Some activity was also demonstrated in the duodenum, placenta, spleen, Peyer's patches, glandular stomach, and jejunum, but no bioactivity was present in other gastrointestinal, endocrine, lymphoid, or haematopoeitic tissues. We were also unable to detect any LAF activity in the reproductive organs (except for the testis), urinary tract, skeletal and muscular tissues, brain, eyes, salivary glands, or lung. In the esophagus the activity was mainly localized to the mucosa. The LAF activity in the skin was partly inhibited by treatment with a mixture of antibodies against human IL-1 alpha and IL-1 beta. Dose response curves and gel filtration on a Sephacryl S-200 column suggested the presence of a high molecular weight (90,000-100,000 Da) LAF inhibitory factor in the liver. In all positive tissues, the demonstrated LAFs had a molecular weight of 15,000-25,000 Da, as determined by Sephacryl S-200 gel filtration. Of the positive tissues, the skin, tongue, esophagus, and the proventricular part of the stomach all contain stratified squamous epithelium. It is tempting to suggest that the detected LAFs have a similar function in these barrier tissues, e.g., to serve as host defence factors, or, alternatively or additionally, as tissue growth factors.     

Characterization of lymphocyte-activating factor (LAF) produced by a macrophage cell line, P388D1. II. Biochemical characterization of LAF induced by activated T cells and LPS.

Unstimulated P388D1 cells, as well as P388D1 cells stimulated with PHA-activated guinea pig T lymphocytes or LPS, produced a lymphocyte activating factor (LAF). In order to have a chemical basis for comparing this LAF with the LAF produced by normal macrophages, we have analyzed several biochemical characteristics of the P388D1-derived LAF. Sephadex G-75 chromatography of concentrated LAF-containing supernatants from cultures of unstimulated and T cell stimulated P388D1 cells demonstrated that the cell line LAF had a m.w. of approximately 16,000. On DEAE cellulose, the T cell-induced LAF fractionated into at least three major peaks and one minor peak. By using hydroxylapatite chromatography, two of the major peaks of LAF activity were separated from residual contaminating Lowry positive material. LPS-stimulated P388D1 also produced LAF with a m.w. of 16,000. However, the LPS-induced LAF appeared to lack one of the DEAE peaks of LAF activity observed with the T cell-derived LAF. In contrast to LPS, T cells may induce the synthesis and/or release of an additional LAF component or enzymatically modify one or more of the LAF species that are produced in response to both stimulants. Based on the results of chemical characterization studies, the P388D1-derived LAF appears to be similar in size and charge to the lymphocyte activating factor produced by normal macrophages.     

The role of subcellular factors in pulmonary immune function: physicochemical characterization of two distinct species of lymphocyte-activating factor produced by rabbit alveolar macrophages

T cell stimulatory factors produced by rabbit alveolar macrophages were investigated. Physicochemical characterization revealed that alveolar macrophages (harvested by bronchopulmonary lavage and stimulated in tissue culture with bacterial lipopolysaccharide) released 2 predominant species of lymphocyte-activating factor (LAF) with isoelectric points of 4.6 and 7.4, and m.w. of 14,400 and 11,600 daltons, respectively, as calculated by the Svedberg equation. Using C3H/HeJ mouse thymocytes (and in some instances nylon wool-purified nonadherent rabbit spleen or lymph node cells) as target cells, rabbit LAF was found to induce proliferative responses directly, as well as enhance proliferative responses to phytomitogens. Both LAF species were inactivated by heating, treatment with trypsin, or at low (2.3) pH. The pI 7.4 LAF was also unstable at high pH (9.0). The thymocyte stimulatory activity of both LAF species was not inhibited by the anti-proteases alpha-1-anti-trypsin, Traysylol (aprotinin), leupeptin, or pepstatin.     

The biological activity in vivo of recombinant murine interleukin 1 in the rat

The present study summarizes the biological response of rats to infusion with recombinant murine IL-1 (rIL-1) cloned in Escherichia coli. Thirty-seven male rats (135-180 g) were infused over a 6-hr period with either 0.008 M guanidine hydrochloride (the vehicle) or E. coli product (both groups are controls) or 1000, 3750, 7500, 15,000, or 37,500 LAF units/hr of rIL-1. The controls and the group receiving 1000 LAF units/hr of rIL-1 did not exhibit a change in body temperature during the experiment. A mild fever was noted with 3750 LAF units/hr which became significantly elevated with 7500 and 15,000 LAF units/hr. At a dose of 37,500 LAF units/hr of rIL-1 (in 0.08 M guanidine hydrochloride) the rats became hypothermic and died. An equivalent dose of guanidine hydrochloride alone (0.08 M) was not fatally toxic although the rats did become hypothermic. Plasma zinc levels were significantly depressed and white blood cell count elevated at 6 hr postinfusion onset. Resting energy expenditure (REE) was significantly depressed during an infusion of 7500 and 15,000 LAF units/hr of rIL-1 despite a concurrent elevation in body temperature. Whole-body leucine kinetics were unchanged by infusion with rIL-1. Plasma fibrinogen and serum haptoglobin and copper levels were not altered by rIL-1. In conclusion, murine rIL-1 is similar to monocytic-derived IL-1 in that it produces a fever, hypozincemia, and leukocytosis; however, rIL-1 does not induce changes in protein metabolism.     

Comparative study of splenic natural killer cells and prothymocytes

An increase in the concentration of the acute-phase reactant, serum amyloid A (SAA), following endotoxin treatment, is a consequence of the action of lipopolysaccharide (LPS) on macrophages to produce a monokine, the SAA inducer, which in turn, triggers SAA synthesis by hepatocytes. We have found that murine SAA inducer is closely related, if not identical, to murine lymphocyte activating factor (LAF), otherwise known as Interleukin 1 (IL 1). Furthermore, both rabbit endogenous pryrogen (EP), which is believed to be identical to LAF (IL 1), and human LAF (IL 1), induced elevated SAA concentrations in C3H/HeJ mice. Antiserum previously shown to block both pyrogenic and thymocyte proliferating activities of the species of rabbit EP exhibiting an isoelectric point of pH 7.3 (EP 7), also blocked the SAA inducing activity of EP7. Phenylglyoxal treatment of highly purified murine LAF (IL 1) abrogated both thymocyte proliferating activity and the SAA inducing activity. These studies support and extend previous reports suggesting that within 2 hr of an inflammatory stimulus, macrophages produce a monokine that acts systemically to alter body temperature, activate T cells, and induce hepatic protein synthesis of acute-phase reactants.     

Relationship between production and release of lymphocyte-activating factor (interleukin 1) by murine macrophages. 1. Effects of various agents

The relationship between the production and release of lymphocyte-activating factor (LAF, or interleukin 1) by cultured murine peritoneal macrophages (Mφ) was investigated. Unstimulated Mφ produce high levels of intracellular LAF within a few hours after culturing, but release little of this activity into their culture medium. Addition of various agents was found to increase significantly the production and release of LAF, with three different patterns: (a) Both intracellular and extracellular LAF activities were increased in response to latex beads, (b) A marked increase of intracellular LAF, with just a minimal elevation of extracellular activity, was stimulated by LPS. (c) Sharp increases in LAF release, with small increments of the intracellular activity, were induced by silica and glucocerebroside (GL₁). Silica and GL₁ damaged the cultured Mφ, as indicated by the increased release of lactate dehydrogenase. It is significant, therefore, that silica and GL₁ increased both intracellular and extracellular LAF levels, suggesting that damage of Mφ may stimulate total LAF production. A combination of LPS with silica or GL₁ acted synergistically on Mφ to release very high levels of LAF, which far exceeded those released by the individual agents. The agents were also tested on Mφ which were precultured, to deplete their LAF content. Latex, LPS, or silica increased LAF production and release by precultured Mφ, but the levels were lower than those obtained with freshly cultured Mφ. The results of this study thus show that the level of LAF release does not necessarily reflect the level of total LAF production by cultured Mφ and suggest that injurious agents may promote LAF production.     

Isolation and identification of separated messenger RNAs for rabbit alpha and beta globin.

Rabbit globin messenger RNA was separated into two species by polyacrylamide gel electrophoresis in formamide. The two species were isolated from the gel and assayed for messenger activity in the ascites cell-free system. The product of the cell-free system was analysed by column chromatography and by finger-printing. The RNA species with the lower mobility (mol. wt 227,000) codes mainly for β globin, whilst the RNA with the higher mobility (mol. wt 202,000) codes mainly for α globin. Fingerprint analysis of DNA copies of the separated mRNA species may be distinguished, and suggest that the polynucleotide sequences are of homogeneity comparable to the messenger activity. We conclude therefore that a physical separation of the two messenger species has been obtained.     

THE ACTIVITIES OF BUTYRYLCHOLINESTERASE AND CARBONIC ANHYDRASE, THE RATE OF ANAEROBIC GLYCOLYSTS, AND THE QUESTION OF A CONSTANT DENSITY OF GLIAL CELLS IN CEREBRAL CORTICES OF VARIOUS MAMMALIAN SPECIES FROM MOUSE TO WHALE

Abstract— The question of a constant density of glial cells in mammalian cerebral cortex regardless of species was examined by surveying the cortical activities of two enzymes primarily localized to dial cells. The cortical activity of butyrylcholinesterase (EC 3.1.1.8) was essentially constant at a rate of approx. 0.1 μmol of butyrylthiocholine hydrolysed min^-1 g^-1 over the range of species from rat (brain wt., 1.6 g) to fin whale and sperm whale (brain wt., 6800 and 7800 g, respectively). Over the same range the activity of cortical acetylcholinesterase, a neuronal enzyme, decreases by a factor of 7. Thus, butyrylcholinesterase ranged from < 2 per cent (in small rodent brains) to approximately 10 per cent (in whale brain) of the cortical acetylcholinesterase activity. The cortical activity of carbonic anhydrase (EC 4.2.1.1) was constant at a rate of 6.2 (± 0.25) μmol of CO₂ evolved min^-1 g^-1 over the range of species from guinea-pig (brain wt., 4.75 g) to fin whale (brain wt., 6800 g). These data obtained by assaying the dehydration reaction were confirmed by limited assays of the esterase activity of the enzyme (with p-nitrophenylacetate as substrate) and agreed with limited, previously reported data for the hydration reaction. Thus, the circumstantial evidence strongly favoured a relative constancy of cortical glial cell density regardless of species. The rates of anaerobic glycolysis in the cerebral cortex of various species were also investigated. For six species from mouse (brain wt., 0.4 g) to beef (brain wt., 380 g) cortical anaerobic glycolysis varied only slightly in the range of 50–62 μmol of CO₂ evolved h^-1 g^-l, whereas cortical oxygen consumption for the same range of species decreased by a factor of 3. Previously frozen samples of beef cortex glycolysed at 35 per Cent of the rate of fresh (unfrozen) samples. Since identical rates were obtained for previously frozen samples of fin whale cerebral cortex, we concluded that the relative constancy of cortical anaerobic glycolysis could be extended to the range from mouse to whale and that this aspect of cortical metabolism is probably primarily glial in localization. Some implications of the latter conclusion for the proposed role of astrocytes as modulators of neuronal activity have been discussed.     

Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases

With a procedure that allows the renaturation of the DNA polymerase catalytic activity in situ after SDS-polyacrylamide gel electrophoresis, we have compared the active polypeptides present in extracts from organisms covering a wide evolutionary range from prokaryotes to eukaryotes, namely: Escherichia coli, Oryza sativa, Daucus carota , Neurospora crassa, Dictyostelium discoideum, Saccharomyces cerevisiae, Ceratitis capitata, Leucophaea maderae , Xenopus laevis, rat tissues and human lymphoblastoid cells. Two main clusters of active peptides are visible in mammalian and adult insect tissues, characterized by a mol. wt. greater than 70000 and less than 50000, respectively. High mol. wt. peptides are heterogeneous in size and correspond to active fragments of DNA polymerase alpha, whereas low mol. wt. peptides show the same migration rate as purified DNA polymerase beta and are not generated by proteolysis of the high mol. wt. cluster, In the three species of fungi studied, only high mol. wt. peptides are found. The same is true in plant cells, where no DNA polymerase beta activity is detectable and the pattern of the high mol. wt. cluster is similar to that observed in E. coli extracts (which also lack low mol. wt. peptides). Also in mitochondria from higher and lower eukaryotes only high mol. wt. species are observed, and the active band(s) range from 70000 to 145000 daltons. Our results indicate that the structure of DNA polymerase has been highly conserved during evolution so that an active fragment of mol. wt. greater than or equal to 70 000 is always found in prokaryotic enzymes and in the replicative species of eukaryotic and mitochondrial DNA polymerases; at a certain stage in evolution, another species of low mol. wt. DNA polymerase (beta or beta-like) appears.     

Evolutionary trends of neurofilament proteins in fish.

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.     

Interleukin 1 production by the human monocyte cell line U937 requires a lymphokine induction signal distinct from interleukin 2 or interferons

A soluble product from cloned human T lymphocytes is capable of stimulating U937 cells, a line of human monocytes, to produce interleukin 1 (IL 1). We previously reported that U937 cells exposed to T lymphocyte-conditioned medium secrete mononuclear cell factor (MCF), which increases collagenase and prostaglandin E2 production by adherent rheumatoid synovial cells. Whereas structural and functional homologies between lymphocyte-activating factor (LAF, or IL 1) and MCF were described, previous attempts to measure LAF secretion by lymphokine-stimulated U937 cells were unsuccessful. Although the crude supernatants of cultured U937 cells exposed to medium from lectin-stimulated peripheral blood or cloned T lymphocytes contained MCF activity, no LAF activity was detected. After these crude supernatants were chromatographed on Ultrogel AcA54, however, and the fractions were individually assayed for IL 1, MCF and LAF activities were coeluted with apparent m.w. approximately 14,000 to 23,000. The inability to detect LAF activity in the unfractionated medium was accounted for by an inhibitor of lymphocyte proliferation present in fractions of higher m.w. The T lymphocyte product that stimulated U937 cell maturation and monokine production was secreted in response to lectin-stimulation in a dose-dependent fashion. Although we have previously demonstrated that the hormone 1,25-dihydroxyvitamin D3 caused maturational changes in U937 cells, and other investigators have reported effects of alpha and gamma interferon, these changes are dissociable from IL 1 production. Thus, a distinct lymphocyte-derived signal, necessary for the production of IL 1 by U937 cells, can be identified and dissociated from other biologic products that cause "maturational" changes. The detection of LAF activity in U937 cell supernatants requires the removal of an inhibitor of lymphocyte proliferation.     

Cleavage of bacterial flagellin with cyanogen bromide. Antigenic properties of the protein fragments

1. Four polypeptide fragments, obtained by cyanogen bromide treatment of the protein flagellin from Salmonella adelaide, were tested for their antigenic activity by using them as inhibitors in three different assays: bacterial immobilization, haemagglutination of sensitized erythrocytes and quantitative micro precipitation. Immunodiffusion studies were also performed on the protein fragments. 2. Cleavage of the flagellin molecule in this way gave no detectable loss of antigenic determinants. Fragment A (mol.wt. 18000), the largest of the polypeptides, contained all the antigenic specificities present on flagellin that were recognized by the antisera used. In one test, fragment B (mol.wt. 12000) also contained antigenic activity to an extent not easily explainable by contamination with fragment A. Fragments C (mol.wt. 5500) and D (mol.wt. 4500) appeared to be antigenically inactive.     

Monokine‐like activities released from a chicken macrophage line

Abstract

HD11 macrophages were stimulated with Staphylococcus aureus and supernatants were found to have lymphocyte activation factor (LAF), cartilage resorption (CR) lipoprotein lipase inhibition (LPLI), skeletal muscle catabolic (SMC) and cytotoxicity (CTX) activities. Fractionation of crude supernatants by DEAE ion exchange chromatography resulted in 4 peaks of LAF activity, two of which also contained substantial CR activity. Two additional peaks were resolved that had CR activity but little LAF activity. The high pI peak of LAF and CR activity also possessed LPLI, SMC and CTX activity. Further purification of the high pI peak from DEAE by molecular sizing chromatography resulted in a single peak of LAF activity which also contained CTX and LPLI activity. This study indicates that chicken macrophages release monokines similar in activity to mammalian interleukin 1, tumor necrosis factor and/or interleukin 6.     

Controlled release of water-soluble polymeric complexes of sorbic acid with antifungal activities

We synthesized six water-soluble polymeric complexes of sorbic acid with polyvinylpyrrolidone of different molecular weight (mol wt). As shown by infrared absorption spectrum analysis, the complexes were formed by hydrogen bonding. The complexes (SC1, with mol wt=10 kDa, SC2 with mol wt=25 kDa, SC3 with mol wt=30 kDa, SC4 with mol wt=40 kDa, SC5 with mol wt=90 kDa, and SC6 with mol wt=360 kDa) were characterized as low mol wt (SC1, SC2, and SC3) and high mol wt (SC4, SC5, and SC6). The antifungal potencies of the complexes were tested by the macrodilution susceptibility method against environmental and clinically important fungi. Sorbic acid as well as the complexes exhibited minimum inhibitory concentrations (MICs) lower than potassium sorbate against all the strains tested. MICs of SC1, SC2, and SC3 were shown to be 2- to 4-fold lower for yeast and 1.5- to 3-fold lower than those of sorbic acid for moulds, respectively. The MICs of SC4 and SC5 against both of the Candida species tested ranged from 500 to 800 microg/ml, whereas for SC6 and sorbic acid they were about 1 mg/ml. The potencies of the high mol wt complexes against moulds were decreased by increasing the mol wt. For both of the moulds tested, the MICs of SC4 were slightly lower than those of sorbate. The MICs of sorbic acid and SC5 were equal to 300 microg/ml and 500 microg/ml respectively for Aspergillus parasiticus and for Penicillum viridicatum. The susceptibility to SC6 of all of the hyphomycetes tested was higher than that to sorbic acid. The low mol wt complexes and the sorbic acid exhibited minimal fungicidal concentrations (MFCs) 2 and 3 times higher respectively than the MICs. Sorbic acid and SC3 at a concentration of 2.5 mg/ml in an in vitro time kill curve study of Candida tropicalis were shown to be fungistatic, whereas SC1 and SC2 were fungicidal at the same concentrations. For Aspergillus parasiticus sorbic acid at 2.5 mg/ml was fungistatic for a 24-h period, whereas SC1, SC2, and SC3 were fungicidal.     

Interleukin 1 is present in normal human epidermis

We investigated the presence of interleukin 1 (IL 1)-like molecules in normal unstimulated human epidermal tissue. Epidermis from 21 healthy individuals that was prepared by two different methods showed prostaglandin E2 (PGE2) and collagenase stimulating activity for human dermal fibroblasts. All epidermal extracts tested were positive for thymocyte comitogenic activity (lymphocyte activating factor; LAF). Removal of the horny layer decreased epidermal IL 1-like activity. In contrast to epidermal tissue, freshly isolated peripheral blood mononuclear cells (PBMC) contained no detectable PGE2 stimulatory activity. They could, however, produce PGE2 stimulatory activity after culture and stimulation with phytohemagglutinin (PHA) and concanavalin A (Con A). Little membranous IL 1-like activity could be detected in epidermal extracts when using a method that has previously rendered membranous IL 1 from murine proteose peptone-elicited peritoneal macrophages. Gel filtration chromatography yielded double peaks at m.w. approximately 30,000 and approximately 17,000 for all three activities. High pressure liquid chromatography (HPLC) analysis identified two species with a m.w. of approximately 17,000, and one approximately 30,000 species nondissociable in detergent, all having superposable PGE2 and collagenase stimulatory as well as LAF activity. These results establish the existence of IL 1-like molecules, together with a possible precursor, in normal human epidermis. The release of these preformed epidermal IL 1 stores might be important in vivo.     

Comparison of the substrate specificities of protein phosphatases involved in the regulation of glycogen metabolism in rabbit skeletal muscle.

Muscle extracts were subjected to fractionation with ethanol, chromatography on DEAE-cellulose, precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200. These fractions were assayed for protein phosphatase activities by using the following seven phosphoprotein substrates: phosphorylase a, glycogen synthase b1, glycogen synthase b2, phosphorylase kinase (phosphorylated in either the alpha-subunit or the beta-subunit), histone H1 and histone H2B. Three protein phosphatases with distinctive specificities were resolved by the final gel-filtration step and were termed I, II and III. Protein phosphatase-I, apparent mol.wt. 300000, was an active histone phosphatase, but it accounted for only 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities and 2-3% of the phosphorylase kinase phosphatase and phosphorylase phosphatase activity recovered from the Sephadex G-200 column. Protein phosphatase-II, apparent mol.wt. 170000, possessed histone phosphatase activity similar to that of protein phosphatase-I. It possessed more than 95% of the activity towards the alpha-subunit of phosphorylase kinase that was recovered from Sephadex G-200. It accounted for 10-15% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activity, but less than 5% of the activity against the beta-subunit of phosphorylase kinase and 1-2% of the phosphorylase phosphatase activity recovered from Sephadex G-200. Protein phosphatase-III was the most active histone phosphatase. It possessed 95% of the phosphorylase phosphatase and beta-phosphorylase kinase phosphatase activities, and 75% of the glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities recovered from Sephadex G-200. It accounted for less than 5% of the alpha-phosphorylase kinase phosphatase activity. Protein phosphatase-III was sometimes eluted from Sephadex-G-200 as a species of apparent mol.wt. 75000(termed IIIA), sometimes as a species of mol.wt. 46000(termed IIIB) and sometimes as a mixture of both components. The substrate specificities of protein phosphatases-IIA and -IIB were identical. These findings, taken with the observation that phosphorylase phosphatase, beta-phosphorylase kinase phosphatase, glycogen synthase phosphatase-1 and glycogen synthase phosphatase-2 activities co-purified up to the Sephadex G-200 step, suggest that a single protein phosphatase (protein phosphatase-III) catalyses each of the dephosphorylation reactions that inhibit glycogenolysis or stimulate glycogen synthesis. This contention is further supported by results presented in the following paper [Cohen, P., Nimmo, G.A. & Antoniw, J.F. (1977) Biochem. J. 1628 435-444] which describes a heat-stable protein that is a specific inhibitor of protein phosphatase-III.     

Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli

The high-level expression of human interleukin-1 beta in Escherichia 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 vitro 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-1/LAF assays, are 2 X 10(7) and 4 X 10(7) units mg-1, respectively.     

Contents and morphological distribution of 2,4-dihydroxy-l,4-benzoxazin-3-one and 2-benzoxazolinone in Acanthus mollis in relation to protection from larvae of Pseudaletia impuncta

Secondary metabolites 2,4‐dihydroxy‐l,4‐benzoxazin‐3‐one(DIBOA) and 2‐benzoxazolinone (BOA) were quantified in different morphological parts of A. mollis. The highest DIBOA content was determined in the stamens of the flowers (57.0 umol g^?1 fr. wt). Content of DIBOA in leaves decreased from approximately 28 μmol g^?1 fr. wt for younger leaves (less than 4 wk old), to 3.0 μmol g^?1 fr. wt. for the older ones (more than 13 wk of age). The concentration of BOA was lower than that of DIBOA in all parts of the plant (less than 1.5 μmol g^?1 fr. wt) and showed a small variation. Younger leaves exhibit antifeeding activity against the larvae of Pseudaletia impuncta a native moth that feeds on cereals. These results suggest that DIBOA protects A. mollis species from phytophagous insects.