Alterations of polymorphonuclear leukocyte surface charge by stimulated lymphocyte supernatants

The effects of human lymphokines on the surface charge density of human polymorphonuclear (PMN) leukocytes have been determined using the laser Doppler technique of electrophoretic light scattering. Unfractionated antigen (streptokinase-streptodornase or candida)-stimulated lymphocyte supernatants were found to decrease the mode electrophoretic mobility by 14%. In order to identify the responsible factor, we subjected supernatants from concanavalin A-stimulated lymphocytes to gel filtration on Sephadex G-100 columns and assayed the fractions for their ability to alter PMN electrophoretic mobilities. Two distinct species in the molecular weight ranges of 30–60K and 10–20K, respectively, were found to decrease the electrophoretic mobilities of PMN leukocytes. We have observed no effect of leukocyte inhibitory factor (LIF) on the electrophoretic mobility distribution of PMN leukocytes over a varying period of time (0–8 hr) and over a range of 2- to 10-fold supernatant concentration. Pretreatment of PMN leukocytes with neuraminidase substantially reduced their electrophoretic mobility; the addition of LIF to these pretreated cells did not alter their electrophoretic mobility distribution further. The latter finding is particularly significant in view of the fact that neuraminidase pretreatment of the target cells is known to potentiate LIF activity. We conclude that the mechanism of the inhibition of leukocyte migration by LIF does not involve an alteration of the leukocyte surface charge density.     

Amplification of the activity of human leukocyte inhibitory factor (LIF) by the generation of a low molecular weight inhibitor of PMN leukocyte chemotaxis

Leukocyte inhibitory factor (LIF), which was derived from human peripheral blood lymphocytes by stimulation with concanavalin A ad partially purified by Sephadex G-100 gel filtration, inhibited the in vitro spontaneous migration and chemotaxis of human PMN leukocytes as assessed in a Boyden chamber micropore filter assay. The inhibitory activity was attributed to LIF, a principle defined in terms of its inhibition of PMN leukocyte migration from glass capillary tubes since it was preferentially directed to PMN leukocytes as compared to mononuclear leukocytes, exhibited a size comparable to LIF by gel filtration, and was inactivated by diisopropyl fluorophosphate in parallel with LIF. Incubation of PMN leukocytes with LIF released additional inhibitory activity, distinct from LIF, which resembled the neutrophil-immobilizing factor (NIF) by virtue of its approximate m.w. of 4000 by filtration on Sephadex G-25, inactivation by trypsin digestion, and preferential noncytotoxic inhibition of spontaneous migration and chemotaxis of PMN leukocytes as compared to mononuclear leukocytes. Thus LIF inhibits PMN leukocyte migration both by a direct action on the cells and by an amplification pathway that is mediated by low m.w. chemotactic inhibitors similar to NIF.     

Effect of different leukocyte pyrogen fractions on hemopoiesis

Fractionation of leukocyte pyrogen on a column of Sephadex G-75 made it possible to obtain separately the fraction stimulating the hemopoiesis and the fraction possessing the pyrogenic activity and inhibiting the hemopoiesis. Judging by the elution profile of Sephadex column G-75, substances of high molecular weight produced a stimulating action, and of low molecular weight--pyrogenic and inhibitory action. Possibly pyrogenic and inhibitory activities are connected with different substances. The nature of the inhibitory factor requires further investigation. It may be supposed that it is a substance of chalone type.     

In vivo effects of human dialyzable leukocyte lysate: III. Modulation of the spleen cell proliferative response to antigen by components of leukocyte dialysates and an initial characterization of an ampliative nucleoside

The augmentative effects of isolated components of human dialyzable leukocyte lysates upon the proliferative response to antigen were investigated. Sequential Sephadex G-25 and Bio-Gel P-4 chromatography separated five distinct fractions which, 24 hr after injection into Keyhole limpet hemacyanin (KLH)-sensitive mice, either augmented or suppressed the in vitro spleen cell proliferative response to KLH. An amplifier molecule was isolated from one of the augmentative fractions by high-pressure, reverse-phase liquid chromatography. Preliminary structural analysis of the amplifier component indicated a nucleoside structure, similar to—but possibly distinct from—thymidine.     

Effect of serum fractions from cancer patients on leukocyte migration out of capillary tubes]

Blood serum of patients suffering from cancer of the stomach and urinary bladder inhibited in vitro migration of autologous leukocytes, leukocytes of donors and control patients, and also guinea pig macrophages in over half of cases. In chromatography of these sera on Sephadex G-100 the activity inhibiting the leukocyte migration was revealed in fraction I (Mol. wt. over 100000) and in fractions IV and V (Mol. wt under 30 000). The blood serum and its fractions from cancer patients failed to eliminate the leukocyte migration inhibition caused by the tumour antigens in comparison with the leukocyte migration in the medium with control serum without any antigens. As suggested, the activity of fraction I inhibiting the leukocyte migration was due to the antigen-antibody complex, and of fraction IV and V--to a factor similar by its properties to the factor produced in vitro by lymphocytes stimulated by the antigens or mitogens.     

Chromatographic and enzymatic effects on transfer factor-like activity from human leukocytes and porcine spleen dialysate

1. The effect of dialysable transfer factor (TFd), derived from human leukocytes or porcine spleen cells, was measured using Listeria resistance in mice. 2. The molecular weight range of substance(s) containing TF-like activity is in the less than 3500 MW dialysis fraction on the basis of the capacity of peritoneal macrophages to produce superoxide anion (O2-). This biological activity is removed by heating at 56 degrees C. 3. After Sephadex G-10 chromatography of dialysates the significant activities are found in fractions III and IV of human leukocyte dialysate and in fractions of II and III of porcine spleen dialysate. 4. From enzymatic studies, most of the protective activity of both human leukocyte and porcine spleen dialysate is based on the action of small-molecular weight structures containing peptides and/or polynucleotides.     

Germination inhibitors in the pine seed coat

Inhibitors from (Pinus pinea L.) seed coats were separated using paper chromatography, thin layer chromatography, and a Sephadex G-10 column. The inhibitory activity was resolved into several fractions. One of these behaved similarly to abscisic acid. It has exhibed the same properties as ABA in thin-layer chromatography, paper chromatography, and Sephadex G-10 chromatography and in UV absorption and fluorescence spectra. These germination inhibitors, present in the seed coats, are involved in the regulation of P. pinea seed germination.Abbreviations ABA abscisic acid - TLC thin-layer chromatography     

Growth inhibitors in plasma derived human serum

Summary It was reported previously that plasma derived human serum (PDS) inhibited the growth of cells established from malignant human breast tissues and the MCF-7 cell line but did not inhibit the growth of cells from nonmalignant mammary tissues, including the HBL-100 cell line. Plasma derived human serum was fractionated in the current study by molecular sieve chromatography on Sephadex G-100 in an effort to characterize the factor(s) responsible for inhibition. Plasma derived human serum contained several growth inhibitory fractions, which were designated G-1, G-2, G-3, and G-4. The G-1 was associated with the lipoproteins and immunoglobulins of serum. The lipid portion of G-1 inhibited the growth of both MCF-7 and HBL-100 cells, whereas the protein fraction contained a low activity factor directed against MCF-7 cells only. The G-2 also inhibited MCF-7 cell growth at a low specific activity and was separated in the serum albumin fraction. The MCF-7 inhibitory activity in the G-3 fractions from individual donors fluctuated with the level of activity in the starting sera. The cell specific G-3 components were purified further by Sephadex G-100 superfine chromatography and gel electrophoresis. A tentative molecular weight of 50,000 was assigned to the G-3 inhibitor. The G-4 fraction consisted of small molecular weight materials migrating in advance of the column volume, which inhibited the growth of both cell lines. This investigation was supported by Grant PDT-140 from the American Cancer Society, Inc., and PHS Grant CA30284 awarded by the National Cancer Institute, Bethesda, Maryland.     

The identification of inhibitors in bromelin one-stage irregular antibody screening techniques

Human sera contain bromelin inhibitors in irregular antibody screening techniques. In order to identify and purify these inhibitors, pooled human sera were precipitated with ammonium sulfate, followed by DEAE-cellulose and Sephadex G-150 column chromatography. Proteinase activity and inhibitory activity to bromelin were observed in two protein fractions, these were identified as alpha 2-macroglobulin and the S-2 thiol proteinase inhibitor.     

Antioxidant Properties of the Peptides Isolated From Ganoderma lucidum Fruiting Body

Ganoderma lucidum is widely used as traditional medicine for centuries particularly in China, Japan and Korea. Many bioactive metabolites isolated from G. lucidum were therapeutically active against various diseases. The peptide isolated from water extract of G. lucidum was purified by employing Sephadex G-25, Sephadex G-50 and reverse phase HPLC column chromatography. The antioxidant property of the peptide fractions was determined by various in vitro methods. All fractions obtained from Sephadex G-25 and fraction G from Sephadex G-50 are effective antioxidants and comparably fraction C has the highest antioxidant activity. The molecular weight of purified peptide determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration chromatography and Matrix-assisted laser desorption ionization time-of-flight-mass spectrometer was found to be 2.8, 3.34 and 3.35?kDa respectively. The amino acid composition of the peptide was rich in phenylalanine, aspartic acid, proline, histidine and isoleucine. Peptide isolated in the present investigation suggests that has beneficial antioxidant properties may be due to its low molecular weight and specific amino acid composition.     

Comparison of the binding affinities of rabbit IgG fractions to the rabbit fetal yolk sac membrane: use of 22Na to facilitate quantitation of 125I-IgG binding.

Rabbit IgG was purified, Fr-1-(G-200)2, and then separated by DEAE-cellulose column chromatography into three major fractions, Fr-I, -II, and -III. Binding affinities of the 125I-labeled IgG and its fractions to the rabbit fetal yolk sac membrane were studied. At a concentration of 2 mg/ml, rabbit IgG is bound to the extent of 9 mug/cm2 membrane, whereas the values for fractions Fr-I, -II, and -III are 13, 7, and 5 mug/cm2, respectively. The maximal amount of IgG bound appears to be approximately the same, i.e., 23 mug of IgG/cm2 membrane, for Fr-1-(G-200)2 and its three fractions. In contrast, bovine IgG does not bind to the membrane over the range of concentration tested. Binding constants for Fr-1-(G-200)2, fraction Fr-I, -II, and -III are estimated to be 5.4, 8.6, 4.0 and 2.0 times 10(4) M-1, respectively. The binding affinities of IgG fractions to the yolk sac membrane correlate with the chemical and physicochemical properties of the fractions. Also detailed in the text is the use of 22Na to facilitate quantitation of specific binding of the 125I-IgG to the membrane under equilibrium conditions.     

Purification and identification of novel angiotensin-I converting enzyme (ACE) inhibitory peptides from cultured marine microalgae (Nannochloropsis oculata) protein hydrolysate

Isolation of bioactive compounds and commercialization of marine microalgae sources are interesting targets in future marine biotechnology. Cultured biomass of the marine microalga, Nannochloropsis oculata, was used to purify angiotensin-I converting enzyme (ACE) inhibitory peptides using proteases including pepsin, trypsin, α-chymotrypsin, papain, alcalase, and neutrase. The pepsin hydrolysate exhibited the highest ACE inhibitory activity, compared to the other hydrolysates and then was separated into three fractions (F1, F2, and F3) using Sephadex G-25 gel filtration column chromatography. First fraction (F1) showed the highest ACE inhibitory activity and it was further purified into two fractions (F1-1 and F1-2) using reverse-phase high-performance liquid chromatography. The IC₅₀ value of purified ACE inhibitory peptides were 123 and 173 μM and identified as novel peptides, Gly-Met-Asn-Asn-Leu-Thr-Pro (GMNNLTP; MW, 728 Da) and Leu-Glu-Gln (LEQ; MW, 369 Da), respectively. In addition, nitric oxide production level (%) was significantly increased by the purified peptide (Gly-Met-Asn-Asn-Leu-Thr-Pro) compared to the purified peptide (Leu-Glu-Gln) and other treated pepsin hydrolysate fractions on human umbilical vein endothelial cells (HUVECs). Cell viability assay showed no cytotoxicity on HUVECs with the treated purified peptides and fractions. These results suggest that the isolated peptides from cultured marine microalga, N. oculata protein sources may have potentiality to use commercially as ACE inhibitory agents in functional food industry.     

Allergenic activity of some kinds of plant pollen

Allergenic active fractions of ragweed, wormwood, goosefoot and sunflower pollen with a molecular weight of 37 000, 19 000, 35 000 and 14 000, respectively, were isolated by chromatography on Sephadex. All studied allergens had similar properties: affinity to Sephadex which was more pronounced to Sephadex G-75 than to G-100; absorbing components had no activity; allergenic and antigenic activities were determined only in fractions eluted in the bed volume of the column. Affinity of the allergans to Sephadex did not depend on the eluent type, but decreased in potentiation of the buffer ionic strength.     

Purification and preliminary characterization of human leukocyte elastasel.

Affinity chromatography permits the purification of 1–3 mg of human leukocyte elastase from the leukocytes contained in 500 ml of whole blood. Lysosomal granule proteins are extracted from polymorphonuclear leukocytes and subjected to chromatography on a column of elastin-Sepharose. Contaminating proteins are eluted with buffer containing 1 m NaCl and then elastase activity is eluted with buffer containing 8 m urea. The enzyme retains all of its esterase activity against N-t-BOC-l-alanine p-nitrophenyl ester after exposure to 8 m urea and retains 22% of its activity in the presence of 1% sodium dodecyl sulfate. In sodium dodecyl sulfate and 2-mercaptoethanol leukocyte elastase undergoes autolysis giving rise to several low molecular weight fragments. The molecular weight of the native enzyme is found to be 22.000 by both gel filtration and sodium dodecyl sulfate—acrylamide gel electrophoresis. A characteristic set of four isozymes is seen after acrylamide disc gel electrophoresis at pH 4.5. All bands are active against elastin and also contain carbohydrate by the periodic acid-Schiff stain. On the basis of stain intensity, the slower moving isozymes appear to be richest in carbohydrate. Active leukocyte elastase forms a complex with α₁-antitrypsin in a 1:1 molar ratio. The elastase must be enzymatically active for complex formation to occur.     

Neutral proteinase inhibitors in PMN leukocytes. II. Isolation and characterization of a neutral proteinase inhibitor from porcine PMN leukocytes

An inhibitor of neutral proteinases was purified from porcine PMN leukocytes by gel filtration on Sephadex G-75 superfine and ion-exchange chromatography on Mono S. Thus an inhibitor preparation with a specific inhibitory activity against chymotrypsin of 10 IU/mg was obtained. In dodecyl sulfate gel electrophoresis a single protein band with an apparent molecular mass of 40 kDa was found under reducing conditions. Under non-reducing conditions the inhibitor forms higher molecular mass aggregates. On isoelectric focusing several protein bands with isoelectric points between pH 7.0 and 7.5 could be separated. The amino-acid composition of the inhibitory protein was determined. The inhibition mechanism was studied and association rate constants (kon) were measured and calculated for the reaction with chymotrypsin as well as leukocyte and pancreatic elastase. In Western blot analysis and in enzyme immunoassay studies crossreactivity between antibodies directed against porcine leukocyte neutral proteinase inhibitor and the corresponding inhibitor of bovine PMN leukocytes could be demonstrated.     

Demonstration of an endogenous, competitive inhibitor(s) of [3H] diazepam binding in bovine brain.

An endogenous inhibitor(s) of [³H] diazepam binding to synaptosomes has been demonstrated in bovine brain. The inhibitory activity of crude extracts is heat stable, dialyzable, and not affected by ether extraction. Three distinct peaks of inhibitory activity were resolved using Sephadex G-25 chromatography. The lowest molecular weight peak (<700 daltons) had the highest specific inhibitory activity and its inhibition of [³H] diazepam binding was competitive. A similar low molecular weight fraction was not observed in either muscle or liver suggesting that it may be unique to brain. Thin layer chromatography of the Sephadex G-25 fractions revealed a discrete band of inhibitory activity in the two low molecular weight peaks.     

Ligula intestinalis (Cestoda: Pseudophyllidae): Studies on the properties of proteolytic and protease inhibitor activities of plerocercoid larvae

The somatic extract of L. intestinalis plerocercoids reveals hydrolytic activity against N-Benzoyl-l-tyrosine ethyl ester (BTEE) and Azocoll, and inactivates the esterolysis by mammalian trypsin and chymotripsin. The proteolytic enzyme activity and the inhibitory effect were completely separated by Sephadex G-100 column chromatography. Gel chromatography of the somatic extract revealed two peaks of proteolytic activity : one is bound to macromolecular substances, the other appears to be in free form and has a molecular weight of approx 60,000–65,000. The proteolytic activity showed the following characteristics : Tris-HCl buffer provided the highest activity against BTEE, the pH optimum was 7·4–7·8; the enzyme was activated by 10^?5m-Ca²⁺, Mg²⁺ or Mn²⁺, it was inhibited by 10^?5m-Cu²⁺, but not by 10^?5m-Zn²⁺. 0.001% soybean trypsin inhibitor, 2 × 10^?3m-EDTA, 1 mm-tosyl-l-phenylalanyl chloromethane, 1000 KIU/ml Trasylol did not inhibit the proteolytic activity, but it was inhibited by 1 mm-phenylmethyl-sulphonyl fluoride. The enzyme activity completely ceased upon 5 % TCA treatment or incubation at 56°C for 30 min. The trypsin and chyrnotrypsin inhibitor activities were eluted from the Sephadex G-100 column in a single peak with an estimated molecular weight of 6700–7200. The inhibitory effect was not sensitive to pH changes, and treatment by 5% TCA or incubation at 80°C for 15 min was ineffective. The proteolytic activity of plerocercoid extract was not effected ‘in vitro’ by the inhibitors isolated from this parasite.     

Partial purification and properties of phosphatidate phosphatase in Saccharomyces cerevisiae

Using an aqueous dispersion of [32P]phosphatidate as substrate we detected phosphatidate phosphatase (EC 3.1.3.4) activity in a cell-free extract of the yeast, Saccharomyces cerevisiae. The activity was found in both the membrane and the soluble fractions. The enzyme was purified from the soluble fraction about 600-fold. The purification procedure involved (NH4)2SO4 fractionation, poly(ethylene glycol) 6000 fractionation and column chromatography on DEAE-Sepharose, Sephadex G-100 and Blue-Sepharose. The purified enzyme almost absolutely required Mg2+ for activity. The molecular weight of the enzyme was estimated by analytical gel filtration on Sephadex G-100 to be approx. 75000. The enzyme was highly specific for phosphatidate. The apparent Km for phosphatidate was approx. 0.05 mM. The optimum pH was between 7.0 and 8.0.     

Isolation of an inhibitor of ß-glucuronidase from porcine sublingual gland

An inhibitor of ß-glucuronidase was isolated from porcine sublingual gland by successive fractionation of trypsin extracts of the latter on Sephadex G-100, DEAE-cellulose, Sephadex G-200, and DEAE-cellulose. Its purity and homogeneity were established by DEAE-cellulose column chromatography, ultracentrifugation, and electrophoresis on cellulose-acetate membrane. The sedimentation coefficient of the purified ß-glucuronidase inhibitor was 3.75 S (S₂₀⁰, w), and the molecular weight was determined to be 340 000 from Sephadex G-200 column chromatography. The inhibitor contained 17.5% protein, 20.8% total hexoses, 19.9% hexosamine, 21.8% N-acetylneuraminic acid, and 9.6% fucose. The inhibition was non-competitive, and it was completely suppressed by the addition of NaCl, KCl, Na₂SO₄, or CaCl₂, respectively.     

Elaboration of leukocyte migration inhibitory factor by human lymphocyte subpopulations stimulated with mitogens.

This paper describes experiments to determine whether human lymphocyte sub-populations stimulated with a variety of mitogens, leucoagglutinin (LA), concanavalin A (con A), pokeweed mitogen (PWM), protein A (prot A), and anti-β₂-microglobulin (anti-β₂m), synthesize lymphokines. T and B lymphocytes as well as unseparated mononuclear cells were stimulated with the mitogens, and the presence of leukocyte inhibitory factor (LIF) in the culture supernatants was tested by an agarose migration method. Culture supernatants stimulated with LA or prot A were also fractionated on Sephadex G-100 columns, and LIF-containing fractions were tested for heat stability and the effect of monosaccharides. The results indicated that LA and con A caused LIF synthesis only in T-cell populations, while PWM stimulated both T and B lymphocytes and prot A and anti-β₂mm were B-cell stimulants. Furthermore, LIF from LA-and prot-A-stimulated cultures behaved similarly upon physicochemical characterization.