Dynamic activity of the splenic lymphocytes during the modelling of chronic denervation-delymphatization of the kidneys in mice

In experiments on mice CBA the activity of spleen lymphocytes was investigated in sham-operated mice and in mice with denervation-delymphatization of the both kidneys. The activity of lymphocytes was determined by registration of amounts of colony-formed elements by the method of Till and McCulloch. Periodic increase of the lymphocytes activity has been observed in mice with denervation-delymphatization kidneys. It is supposed that the periodic activation of lymphoid system, induced by denervation-delymphatization, may be one of triggering mechanisms of transplant rejection.     

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.     

Emergence of a surface immunoglobulin recycling process during B lymphocyte differentiation

Surface immunoglobulin (Ig)-mediated endocytosis has been investigated in rat B lymphocytes and plasma cells, using horseradish peroxidase (HRP)-labeled sheep anti-rat Ig Fab' fragment of antibody and HRP as monomeric ligands, respectively. Quantitative estimates of HRP activity associated either with plasma membrane or with endomembrane compartments were made in several experimental conditions. Binding of HRP-conjugate on B lymphocytes was followed by its endocytosis in combination with surface Ig, as shown by the progressive disappearance of plasma membrane-associated HRP activity. Between 1 and 6 h at 37 degrees C in presence of conjugate the total amount of cell-associated activity was constant. These results indicate that during this time no reappearance of surface Ig occurred by neosynthesis, by the expression of an intracellular pool or by the recycling in a free form of the previously internalized molecules. On the contrary, at saturating doses, internalization of HRP by anti-HRP plasma cells increased linearly with time at 37 degrees C in presence of antigen, when, during the same time, the plasma membrane HRP-binding capacity remained constant. Cycloheximide did not affect continuous HRP uptake. The existence of a large intracellular pool of receptors has been ruled out by experiments of removal of binding sites with pronase. In addition, monensin caused a progressive decrease in the number of surface receptors on plasma cells but not on B lymphocytes. Our data then indicate that, unlike B lymphocytes, plasma cells were able to recycle their surface Ig.     

Effects of resveratrol and 4-hexylresorcinol on hydrogen peroxide-induced oxidative DNA damage in human lymphocytes

The protective effects of resveratrol and 4-hexylresorcinol against oxidative DNA damage in human lymphocytes induced by hydrogen peroxide were investigated. Resveratrol and 4-hexylresorcinol showed no cytotoxicity to human lymphocytes at the tested concentration (10-100 μM). In addition, DNA damage in human lymphocytes induced by H 2 O 2 was inhibited by resveratrol and 4-hexylresorcinol. Resveratrol and 4-hexylresorcinol at concentrations of 10-100 μM induced an increase in glutathione (GSH) levels in a concentration-dependent manner. Moreover, these two compounds also induced activity of glutathione peroxidase (GPX) and glutathione reductase (GR). The activity of glutathione-S-transferase (GST) in human lymphocytes was induced by resveratrol. Resveratrol and 4-hexylresorcinol inhibited the activity of catalase (CAT). These data indicate that the inhibition of resveratrol and 4-hexylresorcinol on oxidative DNA damage in human lymphocytes induced by H 2 O 2 might be attributed to increase levels of GSH and modulation of antioxidant enzymes (GPX, GR and GST).     

Indole-3-acetic acid increases glutamine utilization by high peroxidase activity-presenting leukocytes

Indole-3-acetic acid (IAA) is toxic for human tumor cells and in association with horseradish peroxidase (HRP) can be used as a new prodrug/enzyme combination for targeted cancer therapy. The toxic effect of IAA on neutrophils, macrophages and lymphocytes is associated with cell peroxidase activity, which is high in neutrophils and low in lymphocytes. The effect of IAA on glucose and glutamine metabolism in leukocytes presenting different peroxidase activities: neutrophils, thioglycollate-elicited macrophages and lymphocytes was investigated. A time-course effect (from 6 to 48 h in culture) of IAA on glucose and glutamine metabolism of neutrophils, thioglycollate-elicited macrophages, and lymphocytes was then carried out. Addition of IAA (0.25 mM) did not have a marked effect on glucose utilization and lactate formation by the three cell types but it raised glutamine consumption and glutamate production by neutrophils and macrophages. IAA had no effect on glutamine consumption and glutamate production by lymphocytes. A strong relationship was found between glutamine utilization (0.999) and glutamate production (0.999) and peroxidase activity. IAA did not change the activities of hexokinase, glucose-6-phosphate dehydrogenase, citrate synthase, lactate dehydrogenase, and phosphate-dependent glutaminase of 24 h cultured neutrophils and lymphocytes. The effect of IAA (1 mM) on glucose and glutamine metabolism was also investigated by 1 h incubated leukocytes in PBS. IAA did not affect glucose and glutamine metabolism of lymphocytes but enhanced glucose and glutamine metabolism by 1 h incubated neutrophils and thioglycollate-elicited macrophages. IAA caused a marked increase on oxygen consumption by neutrophils, which was more pronounced in the presence of the glutamine as compared to glucose. The stimulation of oxygen consumption leads to a reduction in NADH/NAD+ ratio that activates the flux of substrates through the Krebs cycle. Since glutamine is mainly metabolized through the left hand side of the Krebs cycle, a reduction in the redox state of the cells may accelerate the flux of substrates through glutaminolysis. The toxic results presented here show that the affect of IAA in association with peroxidase involves activation of glutamine metabolism.     

Surfactant–histidine–heme ternary complex as a simple artificial heme enzyme in organic media

A surfactant–heme complex which shows peroxidase activity in organic media has been prepared by a method utilizing water‐in‐oil (W/O) emulsions. Both the aqueous phase pH and the type of surfactant appeared to have prominent effect on the catalytic activity of the heme complex in benzene. The catalytic efficiency of the heme complex was enhanced more than ten times by adding histidine to the aqueous phase of W/O emulsions in the preparation process. The enhancement of peroxidase activity was observed only in a nonaqueous medium due to the increase of the effective concentration of histidine as an activator. In the present study, we propose a simple preparation method for an artificial heme enzyme which works in nonaqueous media. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 502–506, 1999.     

Signal amplification in flow cytometry using biotin tyramine.

BACKGROUND: Catalysed reporter deposition (CARD) has been successfully used as a means of signal amplification in solid-phase immunoassays. The procedure relies on the use of horseradish peroxidase (HRP)-conjugated reagents--normally antibodies-in conjunction with substituted phenolic compounds such as biotin tyramine. The HRP catalyses deposition of biotin tyramine around the site of enzyme activity, and streptavidin-HRP can then be added to generate an amplified HRP signal. The possibility of using this technique for solution-phase amplifications has been suggested but not yet demonstrated. METHODS: This paper describes the application of CARD to signal enhancement in flow cytometry. The specific examples described here are those of anti-human CD4 and anti-human CD36 antibodies binding to either human lymphocytes or mixed mononuclear cells. RESULTS: Optimum biotin tyramine concentrations were evaluated, and a fivefold increase in signal was observed over standard detection of the anti-human CD4 antibody with anti-mouse-fluorescein isothiocyanate (FITC). In the example using the anti-CD36 antibody, the biotin tyramine treatment was repeated, resulting in an additional 2.5-fold signal amplification. CONCLUSIONS: The technique described in this report provides a method of amplifying the signals achieved by standard flow cytometry detection reagents.     

The intensity of lipid peroxidation and enzymatic antioxidative activity in potato leaves under action of drought and polystimulin K

The influence long-term soil drought and potato plants treatment by synthetic analog of cytokinin--polystimulin K on intensity of lipid peroxidation processes and enzymatic antioxidative activity have been investigated. It has been found, that the drought induced the shift of prooxidative-antioxidative balance in respect of lipid peroxidation activation in the potato leaves. It was accompanied by the increase of the ethylene output, membrane permeability, as well as decrease of the lipids content and increase in the enzymatic antioxidative activity (catalase and peroxidase). It is shown, that the intensity of peroxidation processes was higher in budding phases, while enzymatic antioxidative activity was higher in flowering phases in potato plants. Plant exogenous treatment by polystimulin K induced both the decrease in peroxidate oxidation processes, stabilization of catalase and peroxidase activity, as well as the increase in potato resistance to drought.     

Peroxidase activity in the leaf elongation zone of tall fescue : I. Spatial distribution of ionically bound peroxidase activity in genotypes differing in length of the elongation zone

Cessation of cell expansion has been associated with cell wall cross-linking reactions catalyzed by peroxidase. This study utilized two genotypes of tall fescue (Festuca arundinacea Schreb.) that differ in length of the leaf elongation zone to investigate the relationship between ionically bound peroxidase activity and the spatial distribution of leaf elongation. Peroxidase activity was also localized histochemically in transverse sections of the leaf blade using 3,3′ -diaminobenzidine. Soluble or soluble plus ionically bound peroxidase activities were extracted from homogenized segments of the elongating leaf blade and assayed spectrophotometrically. Activity of the ionically bound fraction, expressed per milligram fresh weight or per microgram protein, increased as cells were displaced through the distal half of the elongation zone, corresponding to the region in which the elongation rate declined. In both genotypes, the initial increase in activity preceded the onset of growth deceleration by about 10 hours. In the basal region where elongation began, histochemical localization showed that peroxidase activity was found only in vascular tissues. As cells were displaced farther through the elongation zone, peroxidase activity appeared in walls of other longitudinally continuous tissues such as the epidermis and bundle sheaths. Increase in ionically bound peroxidase activity and changes in localization of peroxidase activity occurred at comparable developmental stages in the two genotypes. The results indicate that cessation of elongation followed an increase in cell wall peroxidase activity.     

Peroxidase activity as a tool for studying the folding of c-type cytochromes

The peroxidase activity of c-type cytochromes increases substantially by unfolding. This phenomenon was used to study the equilibrium unfolding of ferricytochrome c. The peroxidase activity is already enhanced at low denaturant concentrations. The lowest free energy folding intermediate is easily detected by this method, while it is invisible using fluorescence or optical spectroscopy. The free energy difference between this folding intermediate and the native state depends on the strength of the sixth ligand of the heme-iron and the increase in peroxidase activity upon unfolding is shown to be a sensitive indicator of the strength of this ligand. Under fully denaturing conditions, the peroxidase activity is inhibited by protein-based ligands. It is shown that at least three different ligand groups can be responsible for this inhibition, and that at neutral or alkaline pH, the predominant ligand is not histidine. The use of peroxidase activity assays as a method to study the unfolding of cytochrome c is evaluated.     

Estimation of cell surface associated protease activity and its application to lymphocytes

A new method has been developed to estimate proteolytic activity available at the cell surface. Radioiodinated protein substrates are covalently linked to modified polystyrene-divinylbenzene beads with various diameters. These beads are presented to viable cells. Secreted enzyme activity is estimated when no contact occurs between beads and cells. Surface associated proteolytic activity is estimated by the increased rate of iodinated peptide release due to a contact between beads and cells. This method was applied to various lymphocyte preparations. In the absence of serum, mouse spleen lymphocytes produce three- to fourfold higher proteolytic activity than lymph node cells. This activity is completely inhibited by serum diluted 1:10. Since the proteolysis is so marked in the case of spleen cells, one must conclude that lymphocytes removed from the serum and treated in buffered mediums at 37° C have enzymatically altered surface properties. Cell surface associated enzyme activity was measured using rat lymph node lymphocytes with less than 0.1% contamination by granulocytes. This predominantly thymus derived, T cell population had 30% increase in proteolysis due to contact between cells and solid-phase localized substrate of casein. The released enzymatic activity was inhibited by diisopropylfluorophosphate, but its effect on the surface associated enzyme activity remains questionable since it perturbs several membrane functions.     

Influence of salicylic and succinic acids on formation of active oxygen forms in wheat coleoptiles

The comparative study of influence of exogenous salicylic (SaA) and succinic (SuA) acids on the production of reactive oxygen species by isolated wheat coleoptiles has been provided. Under the action of both acids the increase of generation of superoxide anion-radical (O2(.-)) was observed. This increase was partially suppressed by treatment of coleoptiles with inhibitors of peroxidase (salicylhydroxamic acid) and NADP H-oxidase (imidazole and alpha-naphthol). The increase of hydrogen peroxide content, activity of peroxidase and superoxide dismutase (SOD) was registered under the influence of SaA and SuA; catalase activity did not change essentially. The treatment of coleoptiles with the indicated acids resulted in the increase of their resistance to abiotic stress (damaging heating, 43 +/- 0,1 degrees C, 10 min). The conclusion is made, that the increase of O2(.-) generation in wheat coleoptiles under the action of SaA and SuA is related, probably, to the increase of apoplast peroxidase and NADP.H-oxidase activity, and the rise of H2O2 content is related to the growth of SOD activity. These enzymatic systems are involved in the induction of plant cells protective reactions to the hyperthermia.     

Oestrogen as an inhibitor of human NK cell cytolysis

Natural killer (NK) cells are large granular lymphocytes attributed with the ability to lyse certain tumour cells. Previous studies on NK cells have demonstrated only an in vivo suppression of NK cell activity by 17 beta-oestradiol. The suppressive action of oestrogen on other peroxidase-containing leukocytes by virtue of its redox potential has already been documented. In the present study oestrogen suppressed NK cell cytolysis in vitro (determined by the release of [51Cr]chromate from radiolabelled cells) in a dose-dependent manner (p less than 0.01). Parallel experiments demonstrated a similar reduction in NK cell luminol chemiluminescence during activation by K562 tumour cells. Therefore, it would appear that there may be an association between NK cell lysis and their peroxidase/oxygenase activity.     

Homogeneous enzyme immunoassay of diosgenin and its glycosides

Homogeneous enzyme immunoassay has been used as a tool for the determination of diosgenin and its glycosides in plants. Diosgenin antisera was found to inhibit the activity of diosgenin hemisuccinate-horseradish peroxidase conjugate which was reversed by the addition of free diosgenin or its glycosides. The increase of enzyme activity was proportional to the quantity of the hapten over a certain range of hapten concentration. Thus, a minimum of 2.5 micrograms/ml of diosgenin and 11.5 micrograms/ml of diosgenin glycosides could be determined by this method. The results were comparable with those obtained by high-performance liquid chromatography and gravimetric methods.     

IQPA: Isothermal nucleic acid amplification-based immunoassay using DNAzyme as the reporter system

Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.     

A method for large scale purification of turnip peroxidase and its characterization

Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.     

Cytotoxic effect of lymphocytes on autologous blood monocytes in the presence of streptococcal group A antigens in patients with primary erysipelas

The present work deals with a modification of the cytotoxic test for the determination of the cytotoxic activity of lymphocytes in infectious diseases. This modification is based on the use of the suspension of mononuclear blood cells, simultaneously containing effector cells (sensitized lymphocytes) and target cells (autologous monocytes). The cytotoxic effect on monocytes is observed after the preliminary incubation of nonadhering cells (lymphocytes) with the antigen of microorganisms causing the infectious process. A statistically significant increase in the cytotoxic activity of lymphocytes was recorded in patients with primary erysipelas at the acute period of the disease. The cytotoxic effect has been found to persist at a high level for two weeks. By the end of the disease this effect drops to the level characteristic of clinically normal persons. An elevated level of the cytotoxic activity of lymphocytes in the presence of streptococcal antigens of one type has been detected in 72% of patients with primary erysipelas. This indicates that type-nonspecific streptococcal antigens take part in the formation of delayed hypersensitivity, which is also confirmed by the data obtained in animal experiments.     

n-Propanol as a substrate for assaying the ligninperoxidase activity of Phanerochaete chrysoporium.

The steady state kinetics of ligninperoxidase catalysed reaction using n-propanol as the organic substrate and monitoring the formation of propanaldehyde at lambda = 300 nm spectrophotometerically as functions of different reaction parameters has been studied. It has been concluded that n-propanol can be used as a substrate for analysing the activity of ligninperoxidase. The turnover number of ligninperoxidase of Phanerochaete chrysosporium using n-propanol as substrate has been found to be higher approximately by a factor of 10(3) as compared to that using veratryl alcohol as the substrate. The method works in assaying the activity of ligninperoxidase produced by Aspergillus fumigatus indicating that it can be used for assaying the ligninperoxidase activities produced by other microorganisms also and is not limited to assaying the ligninase activity produced by Phanerochaete chrysosporium alone. Under identical experimental conditions, horseradish peroxidase does not show peroxidase activity using n-propanol as substrate indicating that the method does not interfere with the activities of other peroxidases.     

Effect of lipopolysaccharide on the stimulation of macrophage Fc-dependent phagocytosis by splenic B lymphocytes

Macrophage phagocytic activity is regulated by a variety of products derived from activated lymphocytes. It has been reported that nonactivated splenic B and T lymphocytes enhance macrophage glucose metabolism. In addition, the enhancement of macrophage glucose metabolism was further increased by direct effects of bacterial lipopolysaccharide (LPS) on B, but not T, lymphocytes. In the present study, the effect of purified murine splenic B and T lymphocytes on Fc-dependent phagocytosis by thioglycollate-elicited peritoneal macrophages in the presence or absence of LPS has been investigated. Fc-dependent phagocytosis was assayed by measuring the ingestion of 51Cr-tagged sheep erythrocytes. After 3 or 4 days in culture, nonadherent spleen cells (NASC) and B and T lymphocytes from C3H/HeN (LPS-responder) mice produced 92 +/- 27%, 83 +/- 13%, and 147 +/- 33% increases in C3H/HeJ (LPS-hyporesponder) macrophage phagocytic activity, respectively. A similar effect was observed in Balb/c mice. Cell-free supernatant from NASC and B lymphocytes precultured for 2 or 4 days produced a 74 +/- 20% and 157 +/- 42% increase in phagocytosis respectively. At concentrations which have been previously shown to markedly enhance the ability of splenic B lymphocytes to stimulate macrophage glucose metabolism, Escherichia coli K235 LPS (10 micrograms/ml) did not alter the stimulatory effects of any of the splenic lymphocyte populations on macrophage Fc-dependent phagocytosis. These data suggest that B lymphocytes produce a soluble factor(s) which stimulates macrophage phagocytosis. In addition, LPS has different effects on the regulation of macrophage phagocytic activity and metabolism by B lymphocytes.