The current-voltage relationships of liposomes and mitochondria.

Current-voltage relationships were determined for various membrane systems. We show that phospholipid and mitochondrial membranes exhibit linear relations between H+ flux and pH gradients. These membranes, however, exhibited non-linear relationships when the applied voltage was a membrane potential. The current-voltage relationship approximated to an exponential function. This relationship was found to be linearized when the membranes were treated with an electrogenic proton ionophore. The incorporation of cytochrome c oxidase (EC 1.9.3.1) was found to have no effect on the current-voltage characteristics of the phospholipid membranes. When a membrane potential of more than 140 mV was imposed across vesicular and mitochondrial membranes, they exhibited reversible di-electric breakdown. This phenomenon was correlated with the requirement of a permeant ion for the experimental demonstration of proton translocation by so-called 'proton pumps'.     

Nonlinear relationships and phylogenetically independent contrasts

The method of phylogenetically independent contrasts is commonly used for exploring cross-taxon relationships between traits. Here we show that this phylogenetic comparative method (PCM) can fail to detect correlated evolution when the underlying relationship between traits is nonlinear. Simulations indicate that statistical power can be dramatically reduced when independent contrasts analysis is used on nonlinear relationships. We also reanalyze a published data set and demonstrate that ignoring nonlinearity can affect biological inferences. We suggest that researchers consider the shape of the relationship between traits when using independent contrasts analysis. Alternative PCMs may be more appropriate if data cannot be transformed to meet assumptions of linearity.     

Cytokine-induced differentiation of cultured nonadherent macrophages.

Monocytes were isolated from peripheral blood and cultured in vitro for more than 3 weeks in glass chamber slides. Phenotypically and ultrastructurally these nonadherent macrophages (NAM) appear similar to connective tissue resident macrophages. They constitutively secrete a high amount of IL-1ra and little or no IL-1 alpha or IL-1 beta. When exposed to GM-CSF, IL-2, or IFN-gamma for 24 hr, NAM become adherent and undergo dramatic morphological changes. Cytokines treatment primes NAM for increased LPS-mediated TNF production and these GM-CSF- and LPS-treated NAM are cytotoxic to WEHI 164, a TNF-sensitive target. Morphological changes and TNF production are both inhibited by antimetabolites and a variety of antineoplastic drugs. Although morphology inhibition is reversible under certain circumstances, inhibition of TNF synthesis is irreversible. These findings suggest that cytokines might play a role in differentiation and maturation of long-term cultured monocytes. Furthermore, the effects of antimetabolites and antineoplastic drugs on arresting the differentiation processes may significantly impair antitumor functions of macrophages.     

Nonlinear stiffness--force relationships in whole mammalian skeletal muscles

Small, sinusoidal length changes were superimposed on isometric contractions of fast- and slow-twitch mouse muscles, which were stimulated maximally via their nerves. Stiffness increased with increasing frequency of sinusoidal stimulation, but the relative time course of force and stiffness changes during twitch, tetanic, or partially fused contractions was quite invariant over a range of frequencies in both muscles. Typically, stiffness increases more rapidly than force during contraction and decreases less rapidly during relaxation. This pattern was observed at various temperatures and with various numbers of stimuli. It can be described by a nonlinear relation between stiffness and force with some hysteresis. The presence in the muscle of parallel and series elastic elements, whose stiffness varies with force, may account for the nonlinear relation. This nonlinearity can be used to relate the patterns for summation of force and stiffness observed with brief trains of stimuli under a variety of conditions.     

Physical state of cholesteryl esters deposited in cultured macrophages

J774 macrophages load with cholesteryl ester (CE) when incubated with acetylated low-density lipoprotein and cholesterol-rich liposomes; the CE accumulates as cytoplasmic inclusions 1-2 micron in diameter. The CE core of the droplet comprises about 90% of its mass; the predominant CE species present are cholesteryl palmitate (CP, 41%) and cholesteryl oleate (CO, 37%). The thermotropic properties of the inclusions, both in intact cells and after isolation, have been characterized by differential scanning calorimetry. On heating, the inclusions exhibit two endothermic transitions at about 41 and 53 degrees C with a total enthalpy of 7.7 +/- 1.2 cal/g of CE. Very similar thermal behavior is exhibited by a binary mixture containing equal weights of CO and CP; this indicates that these two species dominate the phase behavior of CE in J774 inclusions. A phase diagram for the CO/CP system has been generated, and this reflects simple eutectic behavior. The eutectic is 83% w/w CO, and it melts at 49-50 degrees C. Below this temperature, CO and CP form two immiscible crystalline phases due to the very limited ability of the unsaturated oleate and saturated palmitate acyl chains to mix in the crystal phase. On heating a 1/1 w/w CO/CP mixture, an isotropic liquid of eutectic composition forms at 49 degrees C, and the remaining crystalline cholesteryl palmitate melts over the temperature range 50-69 degrees C. The phase diagram indicates that bulk mixtures of CE molecules in J774 inclusions should be crystalline at 37 degrees C, the growth temperature of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages

Human peritoneal macrophages were cultured in vitro and labeled with [35S]-sulfate. Both on day 1 and day 6 in culture the cells were found to synthesize exclusively chondroitin sulfate proteoglycan, the main part (70%) being associated with the medium after a 20 hour pulse. The glycosaminoglycan chains were found to be oversulfated both after 1 and 6 days in culture, due to the presence of disulfated disaccharide units.     

Transglutaminase and receptor-mediated endocytosis in macrophages and cultured fibroblasts

Summary The function of intracellular transglutaminases remains to be clarified. In fibroblasts the links between the activity of this enzyme and receptor-mediated endocytosis are complex and open to interpretation. However, the issue cannot be firmly laid to rest until the structural specificity of the alkylamine inhibitors of endocytosis is explained. In macrophages, there is substantial evidence that the enzyme plays some role in receptor-mediated phagocytosis, but what this role is and how it might relate to endocytosis in other types of cells is at present an unresolved issue.     

Amelogenins modulate cytokine expression in LPS-challenged cultured human macrophages

Amelogenins are enamel matrix proteins with a proven ability to restore tissues in patients with advanced periodontitis and chronic skin wounds. To explore the mechanisms of action of amelogenins in wound inflammation, the in vitro effect on the expression of selected cell mediators involved in inflammation and tissue repair from human monocyte-derived macrophages was studied. Macrophages were treated with amelogenins in serum-enriched medium with simultaneous lipopolysaccharide (LPS) stimulation, for 6, 24 and 72 h, and the conditioned culture medium was analysed for 28 different cytokines. Amelogenin treatment directed the LPS-induced release of both pro- and anti-inflammatory cytokines towards an alternatively activated macrophage phenotype. This change in activation was also demonstrated by the amelogenin-induced secretion of alternative macrophage activation-associated CC chemokine-1 (AMAC-1, also known as CCL18; p<0.001), a well-documented marker of alternative activation. Amelogenins were also shown significantly to increase the macrophage expression of vascular endothelial growth factor and, to a lesser but significant extent, insulin-like growth factor-1 after 24h of culture. The results of the present in vitro study show that monocyte-derived macrophages stimulated by inflammatory agonist LPS respond to the treatment with amelogenins by reducing the pro-inflammatory activity and increasing the expression of tissue repair mediators.     

Theiler''s virus replication in brain macrophages cultured in vitro.

Infection of the mouse with Theiler's virus is one of the best animal models for the study of multiple sclerosis, a chronic demyelinating disease of the human central nervous system. The identification of the virus target cell(s) is fundamental to an understanding of the viral persistence as well as the inflammation and demyelination observed in the chronic phase of the disease. This paper reports that a small fraction of brain macrophages grown in vitro can be efficiently infected with Theiler's virus without significant cytolytic effect. Viral replication as well as continuous production of infectivity were observed in these cultures.     

Occluding junction structure-function relationships in a cultured epithelial monolayer

Electrical circuit analysis was used to study the structural development of occluding junctions (OJs) in cultured monolayers composed to T84 cells. The magnitude of the increments in transepithelial resistance predicted by such analysis was compared with the magnitude of the measured increments in resistance. Confluent sheets of epithelial cells were formed after cells were plated at high density on collagen-coated filters. Using Claude's OJ strand count-resistance hypothesis (1978, J. Membr. Biol. 39:219-232), electrical circuit analysis of histograms describing OJ strand count distribution at different time points after plating predicted that junctional resistance should rise in a proportion of 1:21:50 from 18 h to 2 d to 5 d. This reasonably paralleled the degree of rise in transepithelial resistance over this period, which was 1:29:59. The ability to predict the observed resistance rise was eliminated if only mean strand counts were analyzed or if electrical circuit analysis of OJ strand counts were performed using an OJ strand count-resistance relationship substantially different from that proposed by Claude. Measurements of unidirectional fluxes of inulin, mannitol, and sodium indicated that restriction of transjunctional permeability accounted for the observed resistance rise, and that T84 junctional strands have finite permeability to molecules with radii less than or equal to 3.6 A but are essentially impermeable to molecules with radii greater than or equal to 15 A. The results suggest that general correlates between OJ structure and OJ ability to resist passive ion flow do exist in T84 monolayers. The study also suggests that such correlates can be obtained only if OJ structural data are analyzed as an electrical circuit composed of parallel resistors.     

Kinetics of high-affinity K+ uptake in plants, derived from K+-induced changes in current-voltage relationships

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV ) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K⁺-H⁺ symporter, responsible for high-affinity K⁺ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K⁺ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K⁺-H⁺ symport-mediated current with a parallel, outward non-linear K⁺ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K⁺ concentration and the cytoplasmic K⁺ concentration. Symport activity is a complex function of the external K⁺ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K⁺ is in the millimolar range and the membrane depolarises. High cytoplasmic K⁺ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K⁺ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed. Received: 23 November 1996 / Accepted: 22 April 1997     

Iron mobilization from cultured rat bone marrow macrophages

The reticuloendothelial system is responsible for removing old and damaged erythrocytes from the circulation, allowing iron to return to bone marrow for hemoglobin synthesis. Cultured bone marrow macrophages were loaded with ⁵⁹Fe-labelled erythroblasts and iron mobilization was studied. After erythroblast digestion, iron taken up by macrophages was found in ferritin as well as in a low-molecular-weight fraction. The analysis of iron mobilization from macrophages shows: (1) the iron was mobilized as ferritin. (2) A higher mobilization was observed when apotransferrin was present in the culture medium. (3) In the presence of apotransferrin in the culture medium, part of the iron was found as transferrin iron. (4) Iron transfer from ferritin to apotransferrin was observed in a cell-free culture medium and this process was temperature independent. The results indicate that after phagocytosis of ⁵⁹Fe-labelled erythroblasts by macrophages, iron is mobilized as ferritin. In the plasma, this iron can be transferred to apotransferrin.     

Heterogeneity among macrophages cultured from mouse bone marrow

Summary The development of macrophages in culture from mouse bone marrow was followed for 14 days by light and electron microscopy, ultrastructural cytochemistry, and flow cytometric analysis. By 10 days greater than 97% of the cells in culture were mononuclear phagocytes, and by 12 days greater than 99% were identifiable as macrophages. Ultrastructurally, three subpopulations of mononuclear phagocytes were distinguished based on the appearance of cytoplasmic structures. Early in culture, cells containing large, membrane-bounded vesicles predominated. With increasing time in culture these cells were replaced to varying degrees first by cells that contained vesicles filled with relatively dense, osmiophilic material and, finally, by macrophages that contained granules of various sizes, shapes and staining densities. Cytochemical (peroxidase and acid phosphatase) and colloidal gold uptake studies at the ultrastructural level suggested that many, if not all, of these cytoplasmic structures arose by pinocytosis and subsequent fusion of pinocytic vesicles with lysosomes. Analysis of DNA content of propidium iodide-stained nuclei by flow cytometry, coupled with the examination of cells treated with colchicine to arrest mitosis in metaphase, suggested that cell cycling was a negligible contributor to heterogeneity within cultured populations. Thus, by waiting until 12–14 days after bone marrow cultures were initiated, with partial replenishment of the culture medium at 7 days, heterogeneity could be greatly reduced in cultured macrophage populations. Taking this fact into consideration could help to reduce the variability seen in functional studies of macrophage populations that are less homogeneous.     

Hyaluronan-binding proteins on cultured J 774 macrophages.

Cultivated macrophages of murine cell-line J 774 were found to bind high-molecular-weight (molecular weight average approx. 5.10(6) [3H]hyaluronan (HA) by a saturable mechanism at 4 degrees C. Half-maximal binding was observed at 7-8 microgram/ml (1.4-1.6 nM) and the maximal binding was reached at 30-40 microgram/ml. Scatchard plot analysis revealed that approx. 20,000 molecules could bind to each cell with a Kd of 1.5 nM. The binding could be effectively inhibited by unlabeled HA. Also chondroitin sulphate inhibited the binding, but only to about 50%. At 37 degrees C the J 774 cells took up and degraded the polysaccharide effectively. Affinity chromatography on HA coupled to agarose of solubilized surface-iodinated J 774 cells, revealed that a protein of approx. 60 kDa, when analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis and autoradiography, could be specifically eluted with HA-oligosaccharides. Our results suggest that J 774 macrophages can bind HA by a mechanism compatible with receptor-binding, and carry a 60 kDa HA-binding protein on their surface. This receptor-binding may mediate uptake and degradation of the polysaccharide and influence the levels and turnover of HA in interstitial fluid as well as the release of HA into the bloodstream.     

Salmonella spp. are cytotoxic for cultured macrophages

We have shown by a variety of microscopical and biochemical techniques that Salmonella spp. are cytotoxic for cultured J774A.1 and bone marrow-derived murine macrophages. The cytotoxicity is initially manifested by inhibition of membrane ruffling and macropinocytosis in infected macrophages, and is followed by cell death. Macrophages killed by Salmonella spp. exhibited features of apoptosis such as condensation and fragmentation of chromatin, membrane blebbing, and the presence of cytoplasmic nucleosomes and apoptotic bodies. Cytotoxicity does not require bacterial internalization as cytochalasin D, a drug that prevents bacterial uptake, did not prevent Salmonella -induced macrophage cell death. However, the cytotoxic effects are strictly dependent upon the expression of the invasion-associated Type III protein-secretion system encoded at centisome 63 of the Salmonella chromosome. Wild-type Salmonella typhimurium grown under conditions that do not allow optimal expression of this system or strains of Salmonella carrying mutations in genes that encode components of this protein-secretion system were devoid of macrophage cytotoxicity. In addition, mutations in invJ , spaO , sipB , sipC and sipD, which encode proteins that are secreted via this secretion apparatus and are required for bacterial entry into non-phagocytic cells, also abolished the toxicity. In contrast, mutations in sipA and sptP , which encode secreted proteins that are not required for bacterial invasion, had no effect on macrophage cytotoxicity. These results indicate a close correlation between the mechanisms of bacterial internalization into non-phagocytic cells and those that lead to macrophage cytotoxicity. Host-adapted serotypes of Salmonella such as S. typhi , S. gallinarum and S. dublin were also toxic for murine macrophages, indicating that this virulence property is probably present in most Salmonella spp. and is not associated with the mechanisms responsible for host range.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.