Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Comparison of the antiviral action of different human interferons against DNA and RNA viruses

Abstract Five different interferon preparations were compared for their antiviral activity against Herpes simplex virus type 1 (HSV-1) and several RNA viruses. The interferons used were: interferon α from human buffy coats, interferon β from human fibroblasts, interferon γ from human lymphocytes after stimulation with phytohemagglutinin (PHA), lymphoblastoid interferon from Namalva cells IFN-α (Ly) and cloned α ₂ interferon produced by Escherichia coli containing the human gene for interferon α ₂. All preparations were able to protect monolayers of HeLa cells against HSV-1 infection when low multiplicities were used. The five IFN preparations were also tested against encephalomyocarditis (EMC) virus, poliovirus and vesicular stomatitis virus (VSV).     

Protective properties of IgM in experiments on animals]

On a model of intraperitoneal infection of albino mice the authors demonstrated a protective action of the fraction enriched with IgM and of gamma-globulin isolated from the normal human blood serum, against E. coli O111. The intensity of the protective action depended on the method, duration of administration of the preparation and also on the infective dose. Protective properties of the fraction enriched with IgM were more pronounced in comparison with the gamma-globulin preparation.     

Phaseolus vulgaris phytohaemagglutinin (PHA) binds to the human T lymphocyte antigen receptor.

The interaction of phytohaemagglutinin (PHA) with the human T lymphocyte antigen receptor (Ti) was explored. Nonidet-P40 lysates of surface-labelled HPB-ALL cells were immunoprecipitated with PHA, using a rabbit anti-(PHA)-serum, as well as clonotypic monoclonal antibodies (H1-2D4 and T40/25) and a rabbit antiserum (R-43) against Ti. One- and two-dimensional SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions showed that both the clonotypic antibodies and PHA precipitated a disulphide cross-linked heterodimer having a mol. wt. of approximately 79 000 (unreduced) and a comprising subunits of mol. wts. approximately 50 000 and 39 000 (reduced). Further evidence that PHA binds Ti was obtained by (i) cross-immunodepletion with H1-2D4 and PHA; (ii) immunoprecipitation with H1-2D4 of a glycoprotein fraction specifically eluted from a PHA immunoprecipitate; (iii) immunoprecipitation with PHA of a solubilised H1-2D4 immunoprecipitate; (iv) 2-D (non-equilibrium pH gradient electrophoresis/SDS) analyses of H1-2D4 and PHA immunoprecipitates, indicated that H1-2D4 and PHA recognise coincident beta polypeptides. PHA also binds a Ti-like disulphide cross-linked heterodimer on tonsil lymphocytes and two other T-cell leukaemias (HUT-78 and J6). The data further suggest that PHA and R-43 recognise a subpopulation of Ti molecules on HPB-ALL cells that are not bound by H1-2D4, suggesting that there may be at least two forms of Ti. Similar experiments indicate that Concanavalin A (Con A) and wheat germ agglutinin (WGA) also probably bind Ti, whereas Helix pomatia agglutinin (HPA) does not.     

Effect of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in autonomic nerves in cat in vitro]

The effects of colchicine and lumicolchicine on the ultrastructure of non-myelinated axons in cat autonomic nerves were studied using in vitro preparations of inferior mesenteric ganglion/hypogastric nerves. After 24 hrs of in vitro incubation with colchicine added to the medium (10 microng/ml) a significant decrease in number of neurotubules per 1 axon was observed. In the presence of a solution of alpha-beta and gamma-lumicolchicine (10 microng/ml) severe degenerative changes occured in axons and Schwann cells. At a lower dose of lumicolchicine (3 microng/ml) these changes were less frequent and the number of neurotubules per 1 axon did not differ from that in control nerves.     

Effect of gamma globulin on reactivity of the organism. V. Types of biological activity of gamma globulin preparations]

Biological activity of 110 series of commercial gamma-globulin preparations was studied; they were found to contain placental antigens, group-specific blood substances, gonadotropic hormones and antibodies to them. Placental antigens were found in 12% of placental and abortive gamma-globulin batches in titres of 1 : 2--1 : 16; no placental protein was revealed in donor gamma-globulin. There were group-specific blood substances in all the batches of placental and abortive gamma-globulin studied (in titres of 1 : 138--A, 1 : 112 B in the placental gamma-globulin and in titres of 1 : 48.9--A, 1 : 32--B in the abortive gamma-globulin). In the preparations from the venous blood group-specific substances were either absent or present in lowe titres only (1 : 2). The value of gonadotropic hormones in the placental gamma-globulin batches constituted 873+/-157, and in the abortive--991.4+/-147 IU/l; no gonadotropins were revealed in donor gamma-globulin. The mean titres of antibodies to gonadotropin hormone in the gamma-globulin preparations made of placental blood constituted 1 : 236+/-32, of abortive--1 : 131+/-16.6, and of the venous blood--1 : 46+/-24.7. The presence of biologically-active substances in the gamma-globulin preparations pointed to the necessity of increased requirement of their quality; additional requirements to its standardization proved to be also necessary.     

Adjuvant actions of polyclonal lymphocyte activators. V. Proliferation of macrophage colony-forming cells in the draining lymph node

We investigated the action of various polyclonal lymphocyte activators (PLA) on the proliferation of macrophage colony-forming cells in vivo at the local site. As PLA, Klebsiella pneumoniae 03 lipopolysaccharide (K03 LPS), Escherichia coli 0111 lipopolysaccharide (E. coli LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemaggulutinin (PHA), polyadenylic-polyuridylic acid (poly(A:U], polyinosinic-polycytidylic acid (poly(I:C], and pokeweed mitogen (PWM) were used. All PLA tested acted to proliferate macrophage colony-forming cells in the draining lymph node at a late stage after subcutaneous injection. The order of strength of this action of PLA was K03 LPS greater than E. coli LPS greater than Con A greater than DS greater than PHA, PWM, poly(I:C), and poly(A:U), which corresponded to the order of strength of their adjuvant action in initiating helper-T-cell response to subcutaneous injection of aggregate-free bovine gamma-globulin. The detailed relationship between the proliferation of macrophage colony-forming cells and the adjuvant action of PLA is discussed.     

Characterization of embryonic liver suppressor cells by peanut agglutinin.

Liver cells of 19-day-old mouse embryos were separated by peanut agglutinin (PNA) into two fractions. The fraction agglutinated with the PNA was found to be enriched for cells capable of suppressing the MLC reaction and the response to the mitogens Con A, PHA, and LPS. The fraction not agglutinated by PNA was significantly less suppressive. The response to DxS was not suppressed by any of these fractions. On the other hand, the response to LPS and DxS, but not to Con A or PHA, was expressed by the nonagglutinated fraction. It is thus inferred that the suppressor cells in the embryonic liver are separable from the potentially reactive cells.     

Evaluation of cytotoxic activity of Vibrio cholerae non-01 culture filtrate on established cell lines and human diploid cells

The cell-destroying effect of cell free filtrates of 90 V. cholerae non-01 cultures was measured by titration method in 3 established cell lines: CHO, HeLa and Vero and in 3 human diploid cells cultures: MRC-5, WI-38 and PZ. The vibrio strains differed in the titre of toxic effect. Most sensitive was CHO cell line, least sensitive were human diploid cell cultures. It was found that bacterial strains produced different substances toxic for various cell lines. Among them NAG-ST toxin produced by 41% of examined strains was identified and hemolysins/cytolysins activity was evaluated. Both may play a role in the pathogenicity of those strains for humans.     

Metabolic changes in wheat (Triticum aestivum L.) plants under action of bioregulator stifun

Under action of growth-stimulating concentrations of bioregulator stifun on wheat plants, an increase of functional activity of nucleoli of meristematic cells; contents of lectin (wheat germ agglutinin); and activity of proteinases, tripsin inhibitors, and ATPase activity was established. The pool of free amino acids was increased under bioregulator use. Levels of methionine, phenylalanine, cysteine, lysine, and tyrosine were increased. It is likely that stifun could activate protein biosynthesis in wheat plants.     

Metabolic changes in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) plants under action of bioregulator stifun

Under action of growth-stimulating concentrations of bioregulator stifun on wheat plants, an increase of functional activity of nucleoli of meristematic cells; contents of lectin (wheat germ agglutinin); and activity of proteinases, tripsin inhibitors, and ATPase activity was established. The pool of free amino acids was increased under bioregulator use. Levels of methionine, phenylalanine, cysteine, lysine, leucine and tyrosine were increased. It is likely that stifun could activate protein biosynthesis in wheat plants.     

DNA synthesis in rat liver following administration of antihepatocytotoxic serum]

Experiments were conducted on adult female rats. The autoradiographic method was applied to the study of thymidine-3H incorporation into the parenchymatous and reticulo-endothelial cells of the liver under conditions of using low doses (0.06 microgram of protein per 100 of body weight) of antihepatocytotoxic serum (AHCS), gamma-globulin isolated from it (gammaAHCS) and gamma-globulin fraction of normal rabbit serum (gammaNRS) to intact animals and rats with carbon tetrachloride affection of the liver. The labelled nuclei index of both the parenchyma and the reticuloendothelial cells increased in case of gammaAHCS administration, and, to a lesser extent, of AHCS to intact animals. gammaAHCS used against the background of CCl4 administration intensified the reparative regeneration. The action of gammaAHCS has phasic character--the period of the labeled nuclei elevation was followed by their reduction, replaced by new intensification of the proliferative processes.     

Agglutinability of cattle red cells. 1. Agglutination by lectins of enzyme-treated red cells

Pronase-treated cattle red cells (CRC) from different monozygous (MZ) twin pairs could be classified as weakly (titre 1: 2, 1: 4) or strongly (titre 1: 1024, 1: 4096) agglutinable when Phytohemagglutinin-M (Phy-M) was used as agglutinin. In the presence of concavalin-A (Con-A), the CRC from different MZ pairs treated with pronase, A-chymotrypsin or trypsin showed a gradation from low (titre 1: 32) to high (titre 1: 256 000) agglutinability. The trypsin-treated CRC which had A₁, A₂ blood factors usually had a titre of 1: 8000 or higher with Con-A. Both the intact CRC and the CRC treated with proteolytic enzymes were capable of absorbing the Phy-M or Con-A lectins.     

Effects of physico-chemical treatments on hemagglutination activity of Anopheles gambiae haemolymph and midgut extract

Abstract. Anopheles gambiae midgut extracts and haemolymph possessed agglutinins, titre 1:16 to 1:256, against human red blood cells (RBCs). Subjection of both tissues to protein precipitation reagents, organic chemical and selected protease, neuraminidase and other glycosidic hydrolase treatments revealed the haemagglutinins to be protein, most likely glycoprotein, in nature - not lipoprotein, lipid, glycolipid or nucleic acid. An.gambiae agglutinins were thermo-labile >40^oC, affected by freezing and thawing treatments, and contained disulphide and hydrogen bonds on the basis of sensitivity following exposure to dithio-threitol and urea respectively. Optimum haemagglutination depended generally on slightly acid to neutral pH conditions and agglutinin activity was Ca²⁺ ion, albeit to a lesser extent Mg²⁺ ion, dependent. The midgut extract agglutinin subunit molecule had a relative molecular weight (M_r) of 65kDa whilst that of haemolymph was 40kDa.
This study presents the first report on selected physico-chemical properties, the glycoproteinaceous nature and tentative subunit M_r of mosquito midgut extract and haemolymph anti-RBC agglutinin(s).     

Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin-1, and epidermal growth factor in human fibroblasts

Tumor necrosis factor (TNF), interleukin-1 (IL-1), and epidermal growth factor (EGF) were mitogenic for human diploid FS-4 fibroblasts. Dexamethasone amplified the growth-stimulating action of all three agents. Amplification of the growth-stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS-4 cells. TNF and IL-1 increased the steady state level of interferon (IFN)-beta 2 mRNA but failed to induce detectable levels of IFN-beta 1 mRNA in FS-4 cells. Dexamethasone inhibited the increase of IFN-beta 2 mRNA levels by IL-1 or TNF. Inhibition of IFN-beta synthesis is likely to be responsible for the inhibition of the TNF-induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN-beta synthesis may also be responsible for the amplification by dexamethasone of the growth-stimulating action of TNF and IL-1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by different mechanism.     

Lectin binding and surface glycoprotein pattern of human macrophage populations

Summary In the present study unstimulated and stimulated human blood monocytes, untreated and phorbol ester treated U-937 cells, as well as human peritoneal and alveolar macrophages were studied with respect to their surface membrane properties. Binding of different lectins and electrophoretic patterns of tritium labeled surface glycoproteins were compared. The analysis of surface glycoproteins could be interpreted as evidence for a common origin of the analysed cell populations. Furthermore, banding patterns of glycoproteins might be useful to define certain activation states within monocyte/macrophage differentiation. In contrast, lectin binding pattern did not clearly discriminate macrophage subpopulations.Abbreviations AM alveolar macrophage - BM blood monocyte - PM peritoneal macrophage - PBS phosphate buffered saline - IPA 12-O-tetradecanoylphorbol-13-acetate - Con A Concanavalin A - HPA Helix pomatia agglutinin - LPA Limulus polyphemus agglutinin - PHA Phaseolus vulgaris agglutinin - SBA Soy bean agglutinin - UEA I Ulex europaeus agglutinin I - WGA Wheat-germ agglutinin     

Effect of staphylococcal toxin and antistaphylococcal gamma-globulin on the electrical and contractile activity of the guinea pig myocardium

A study was made of the action of staphylococcal toxin (ST) and its combination with antistaphylococcal gamma-globulin (ASGG) on intracellular potentials (rest potential--RP, and action potential--AP), and isometric contractions of guinea-pig auricle. ST (initial concentration 18.10(-2)Lh) diluted with normal Tyrode's solution at 1:1000, 1:100 and 1:10 (spontaneously active preparations), and Tyrode's solution with 13.5 mM KCl (evoked activity of preparations), significantly increased the duration of AP of myocardial cells. In evoked activity of preparations, RP and the amplitude of AP declined as the concentration of ST was raised. The amplitude of isometric contractions and maximal rates of their growth and fall increased under the effect of ST (1:1000) and decreased at 1:100 and 1:10. ASGG combined with ST (1:100) did not produce any protective effect on the myocardium. On the contrary, it provoked a still greater inhibition of contractility. The inhibitory action of combined ST and ASGG was seen at all ratios of ST to ASGG (use was made of ASGG shortage, equivalent amount and excess as regards ST) and reached 50% for all study characteristics of contractility. Anatoxin (inactivated toxin) combined with ASGG also produced a cardiodepressant action which was manifested in an approximately 50% decrease in the maximal rate of the growth and fall of contractions in the absence of significant changes in the contraction amplitude.     

Cytostatic and growth-stimulating effects of alveolar macrophages of rats on tumor cells

Cytostatic and growth-stimulating effects of alveolar macrophages (AM) of rats on tumor cells were studied. The experimental results are summarized as follows. 1. The cytotoxicity of AM activated with BCG to tumor cells was increasing with the increase of effector cells/target cells (E/T) ratio. AM without the treatment with BCG expressed slight cytotoxicity to tumor cells at a high E/T, and growth-stimulating effect on tumor cells, at a low E/T. 2. AM after 24-hour culture had a lower manifestation of cytotoxicity to human lung adenocarcinoma cell line than that of AM without 24-hour culture, and had a growth-stimulating effect on B-16 cell line. 3. Cytostatic and growth-stimulating effects of AM without or with 24-hour culture were decreasing with the increase of irradiation doses.     

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.     

Adenylate kinase 2, a mitochondrial enzyme

The subcellular compartmentalization of the isozymes of ATP: AMP phosphotransferase (adenylate kinase) was analyzed in HeLa cells, RAG cells, and RAG-human hybrids that express human AK-2. In HeLa cells and in the hybrids, human AK-2 was present in a mitochondrial fraction prepared from cell extracts and in mitochondria purified by density gradient centrifugation. Human AK-1 was, as expected, distributed in the soluble cytoplasmic fraction of the cells. The rodent isozymes which are homologous to human AK-1 and AK-2 have been determined.This work was supported by NIH Grants HD 04807-07 and HD 06285-04.