Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages

The spectral characteristics of dextran, labeled with fluorescein, depend upon pH. We have loaded the lysosomes of mouse peritoneal macrophages with this fluorescence probe and used it to measure the intralysosomal pH under various conditions. The pH of the medium has no effect on the intralysosomal pH. Weakly basic substances in the medium cause a concentration-dependent increase in the intralysosomal pH. However, the concentration of base necessary to produce a significant change in the intralysosomal pH varies over a wide range for different bases. The active form of the base is the neutral, unprotonated form. Although most of these weak bases cause an increase in the volume of the lysosomes, increase in lysosomal volume itself causes only a minor perturbation of the intralysosomal pH. This was demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a demonstrated in cells whose lysosomes were loaded with sucrose, and in cells vacuolated as a consequence of exposure to concanavalin A. The results of these studies are interpreted in terms of energy-dependent lysosomal acidification and leakage of protons out of the lysosomes in the form of protonated weak bases.     

Effect of number of turbine impellers on surface aeration in laboratory fermentor

The effect of one and two four-flat-bladed turbine impellers on the surface aeration intensity in a laboratory tormentor was investigated at different agitation speeds and sparge rates. The surface aeration was found to be generally more intensive with two impellers for the same other operating conditions because of higher power consumption and the position of the upper impeller closer to the free liquid surface. The surface aeration intensity was successfully correlated in terms of power consumption and sparge rate. The aeration number alone cannot be used for general predicting surface aeration intensities.     

Effect of selenite on cell surface fibronectin receptor

Incubation of cells with selenite, under conditions in which there is no effect on cell viability, results in a decrease in the rate of their subsequent attachment to extracellular matrix proteins such as fibronectin (1). The attachment was inhibited by a pentapeptide containing the RGD sequence and by antibody against the cellular fibronectin receptor (α5β1 integrin), indicating that it is receptor-mediated. To investigate whether exposure to selenite has an effect on fibronectin receptors, we assayed for their presence on the cell surface by measuring the ability of cells to attach to a surface that had been coated with antibodies to the receptor. Brief exposure of cells to low concentrations of selenite resulted in a significant decrease in their ability to attach to monoclonal antibodies against the α5 or β1 subunits of the fibronectin receptor, as well as to polyclonal antibodies against the complete receptor. This indicates that exposure to selenite results in a decrease in receptors that are present at the cell surface. Exposure of the cells to selenate, selenocystine or selenomethionine did not result in a significant decrease in cell surface receptors. Preincubation of the cells with selenite was required for the effect, indicating that selenite does not directly interfere with receptor structure or function.     

Effect of nitrite concentration and pH on most probable number enumerations of non-growing Nitrobacter spp.

Abstract Enumerations of nitrite-oxidizing bacteria in soil samples by a Most Probable Number technique, often showed relatively high cell numbers at a low nitrite concentration compared with the numbers of ammonium-oxidizing bacteria. It was hypothesized that the high numbers enumerated at low nitrite concentration would represent non-growing or organotrophically growing cells of nitrite-oxidizing species. In this paper, the sensitivity of non-growing Nitrobacter species to high nitrite concentrations as well as to low pH was examined. Different Nitrobacter species were pre-cultured at 0.5 mM nitrite. Non-growing cells differing in age were enumerated at different nitrite concentrations and pH values. The incubation period lasted for 5 months at 20°C. However, during the incubation periods of the older non-growing cells, it appeared that a period of 5 months might have been too short for reaching constant numbers. Early stationary cells of all species that were studied appeared not to be affected by high nitrite concentrations or low pH. Eight- and 18-month-old non-growing cells of Nitrobacter hamburgensis were also insensitive to 5 mM nitrite. The numbers of 8- and 18-month-old resting cells of N. vulgaris were only repressed by a combination of 5 mM nitrite and a low pH. Eight-month-old non-growing cells of N. winogradskyi were sensitive to 5 mM irrespective of pH, but 18-month-old cells only to 5 mM nitrate at low pH. The numbers of 8- and 18-month-old resting cells of N. winogradskyi serotype agilis were repressed by low pH rather than high nitrite concentration. Hence, it was concluded that the large differences in numbers of nitrite-oxidizing bacteria obtained with low and high nitrite concentrations in the incubation medium, was not likely to be due to the presence of non-growing Nitrobacter species in soil samples, but rather to the existence of organotrophically growing Nitrobacter cells.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

Summary The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Effect of different fixatives on Con A surface receptors of mouse peritoneal macrophages

The effects of glutaraldehyde, formaldehyde, or osmium tetroxide fixation on the number of labeled Con A surface receptors on mouse peritoneal macrophages were compared. Gold-labeled Con A receptors were found to be isolatedly arranged and evenly distributed on cell surfaces independent of the fixative used. Only cells preincubated with Con A and subsequently fixed by osmium tetroxide showed arrangement of labeled receptors in clusters. Significant differences were found in the number of Con A receptors per cell depending on the fixative used. The fluorescence intensity of FITC-Con A staining was detected spectrophotometrically, the characteristic X-rays of gold-labeled Con A receptors were determined by means of electron beam-induced X-ray microanalysis. The experimental results obtained both at light and electron microscopic level pointed to formaldehyde being the best fixative also for this purpose.     

Effect of the volume of medium and number of oocytes during in vitro fertilization on embryo development in pigs

The present study was designed to determine the effect of the volume of medium (VM) and the number of oocytes (NOOC) during in vitro fertilization (IVF) on embryo development in pigs. Groups of 15, 30 and 50 in vitro matured oocytes were transferred to 2, 1 and 0.1 ml of modified Tris-buffered medium (mTBM) and inseminated with frozen-thawed spermatozoa (2000 spermatozoa/oocyte) in a 3 x 3 factorial experiment. A total of 2739 oocytes from four replicates were exposed to spermatozoa for 6 h and then cultured in embryo culture medium for 6 h (pronuclear formation) or 7 days (blastocyst formation: BF). The efficiency of fertilization (EF: number of monospermic oocytes/total number of inseminated oocytes) and BF decreased (P<0.03) as the VM increased (EF: 45.9+/-2.2, 43.8+/-2.6 and 36.9+/-1.6% and BF: 29.4+/-2.7, 23.2+/-1.8 and 19.9+/-2.1% for VM 0.1, 1 and 2 ml, respectively). The BF, but not EF, was also affected (P<0.04) by NOOC (19.8+/-1.6, 28.1+/-2.3 and 24.6+/-2.9% for groups of 15, 30 and 50 oocytes, respectively). The effect of the interaction VM x NOOC on EF and BF was not significant. These results indicate that when 2000 spermatozoa/oocyte were used, a low volume of IVF medium (0.1 ml) and the number of oocytes during IVF (30-50) can improve the in vitro embryo production in pigs.     

Effect of toll-like receptor activation on thymosin beta-4 production by chicken macrophages

Thymosin beta-4 (Tβ4) is an actin-binding intracellular peptide that promotes wound healing, tissue remodeling, and angiogenesis. The mechanism of Tβ4 secretion to the extracellular environment is not understood. The macrophage is a rich source of Tβ4 which also participates in wound healing process. The objective of this study was to find how Tβ4 may be externalized. Using activation of macrophage through their toll-like receptors (TLR), the changes in cellular Tβ4 was studied. A naturally transformed chicken macrophage cell line HTC was treated with different TLR agonists and the cellular Tβ4 changes was determined at 6 and 24 h after stimulations using stable isotope labelling of amino acids in cell culture (SILAC) and mass spectrometry. Real time PCR was used to determine changes in gene expression. The results showed that TLR agonists such as peptidoglycan (PGN) or lipopolysacharide (LPS) caused depletions in cellular Tβ4 peptide along with its detection in the cell culture supernatant at 24 h. These TLR agonists also induced the expression of interleukins-1β, -6, and nitric oxide synthase genes at 6 h but failed to modulate Tβ4 gene at that time point indicating that the Tβ4 externalization was not associated with its production. To find whether Tβ4 externalization was associated with cell death, we measured the lactate dehydrogenase (LDH) activity of the conditioned media as an indicator of cell damage. The results showed that the TLR agonists which induced depletion of intracellular Tβ4 at 24 h also increased the LDH content of the conditioned media, suggesting that the Tβ4 in the extracellular media most likely originated from dying macrophages.     

Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.     

Stereological estimation of ovarian oocyte volume, surface area and number: application on mice treated with nandrolone decanoate

Changes in the number and size of oocytes can lead to fertilization problems. The present study aimed to evaluate the number, volume, and surface area of oocytes in healthy as well as nandrolone decanoate-treated (ND) mice using stereological methods. Five control mice received vehicle, and five ND-treated mice received ND. Using the 'isotropic Cavalieri' design', the ovary was sectioned. The volume of the ovary (cortex and medulla) was estimated. The oocytes' volume and surface area were estimated using the invariator. The number of the oocytes was estimated using an optical disector. The volumes of the ovary, cortex, and medulla decreased ~50% in the ND-treated mice. The mean number (coefficient of variation) of preantral, antral, and atretic oocytes in the control ovary were 1,690 (0.29), 2,100 (0.52), and 3,900 (0.2), respectively, which decreased ~54%, ~87%, and ~91%, respectively in the ND-treated animals. The mean volume (coefficient of variation) of the preantral, antral, and atretic oocytes were 86,000 (0.27), 110,000 (0.48), and 27,000 (0.33) μm3, respectively. The mean surface area (coefficient of variation) of the three types of oocytes were 9,000 (0.24), 9,900 (0.28), and 4,700 (0.21) μm2, respectively. These parameters remained unchanged in the ND-treated mice. ND induces reduction in the number of oocytes, but not in the volume or the surface area.     

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.     

A lectin-like receptor on mammalian macrophages

Rat Kupffer cells in vitro strongly bind neuraminidase-treated rat erythrocytes but not untreated erythrocytes. Binding between cells is inhibited by preincubation of macrophages with D-galactose and related sugars, but not with unrelated saccharides. We therefore suggest that cell adherence is mediated by a galactose-specific receptor on the Kupffer cell membrane.     

Effect of surface potential on Rb+ uptake in yeast: The effect of pH

The apparent K_m of Rb⁺ uptake and the zeta potential of yeast cells are appreciably affected by changes in the pH, variation of the concentration of the buffer cation Tris⁺ and addition of Ca²⁺ to the suspending medium. Irrespective of the way in which the zeta potential is affected, a direct relationship between the apparent K_m of the Rb⁺ uptake and the zeta potential is observed. A reduction of 8 mV in the zeta potential is accompanied by a 20-fold increase in the apparent K_m, which illustrates that electrostatic effects in ion uptake cannot be ignored. Measured zeta potentials are, to a good approximation, linearly related to surface potentials evaluated from a kinetic analysis of the Rb⁺ uptake. This shows the practical use of the zeta potential as a measure of the surface potential in studies of electrostatic effects in ion uptake by yeast. It is concluded that Tris⁺ and the aikaline earth cations inhibit the Rb⁺ uptake in yeast exclusively via a reduction in the surface potential. Protons, in addition, exert a competitive inhibition.     

Regeneration of plasmalemma and surface properties in macrophages

The regeneration of surface anionic groups in mouse peritoneal macrophages was investigated by electron microscopy, using cationized ferritin (CF) as a tool for the localization and evaluation of negative charge density on the cell surface. In vitro interaction of living macrophages with CF resulted in removal of most anionic groups, either by concentration of their receptor sites to a part of the membrane which is subsequently internalized, or by detachment of the aggregated label from the surface. After incubation of macrophages lacking surface anionic groups in tissue culture medium without the ligand, regeneration of the binding capacity for CF took place within 3 h. The first regenerated parts of the membrane can be visualized within 1 h on the upper part of the adherent cells; there is a discontinuous coating of ferritin, with the lateral regions of the plasmalemma free of label. The attached CF particles on the regenerated membrane are closer to the membrane and their density is considerably higher than on the normal control macrophages. The results indicate that the turnover of the plasmalemma is regional and not dispersed; the mechanism involved is insertion of membrane patches into the pre-existing plasma membrane.     

Polarization of endocytosis and receptor topography on cultured macrophages

In the 1774.2 macrophage cell line, microtubule disassembly by colchicine causes the polarization of membrane functions ane structure. Colchicine-treated cells develop a bulge or protuberance that is bordered by microvillous membrane. The protuberance is the site of concanavalin A cap formation. The fluid pinocytosis of horseradish peroxidase and of fluorescein- and rhodamine-conjugated high molecular- weight dextrans, the adsorptive pinocytosis of concanavalin A, and the concentration and phagocytosis at 37 degrees C of a range of phagocytic particles (IgG- and complement-opsonized erythrocytes, complement- opsonized zymosan, latex shpres, albumin-stabilized oil droplets) are all similarly restricted to the protuberance. A reduction in the rate of dextran pinocytosis, determined by fluorimetry, and reductions in phagocytic rates for oil emulsion and IgG-opsonized erythrocytes accompany the polarization of endocytic activity in colchicine-trated 1774.2 macrophages. Membrane receptors for phagocytic particles are not confined to the protuberance but rather may display their own unique topographical asymmetry. The inherent topography of receptors was inferred from particle distribution under conditions that limit particle-receptor redistribution (after labeling at 4 degrees C or a very brief incubation at 37 degrees C). Under these restrictive conditions, latex binding sites were detected over the whole membrane whereas receptors for IgG-opsonized erythrocytes, aggregated IgG, complement-opsonized erythrocytes, and complement-opsonized zymosan were excluded from the protuberance. Thus, functional (endocytosis) and structural (inherent receptor distribution) analyses of membrane topography define different patterns of asymmetry in protuberant cells. The asymmetry induced in 1774.2 macrophages by colchicine is highly analogous to the functional and structural polarity of epithelial cells. Exploration of this analogy may provide insight into the development of polarized epithelia and, more generally, into mechanisms by which specialized areas of membrane are established.