5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis.
The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface. (+info)
Three-independent-compartment chamber to study in vitro commissural synapses.
We describe a novel chamber in which the two intact neonatal rat hippocampi and the commissural fibers are placed in three independent compartments separated by latex membranes and perfused selectively with different solutions. A set of control tests showed that the compartments are well isolated: 1) methylene blue or eosin applied to one compartment did not diffuse to other compartments when verified via the microscope, and spectrophotometry revealed that <1/10.000th of the dye diffuses to other compartments; 2) tetrodotoxin (1 microM) applied to the commissural compartment blocked the synaptic responses evoked contralaterally without affecting those evoked on the ipsilateral side. This chamber enables a wide range of experiments that cannot be performed in conventional chambers, e.g., to study the maturation and plasticity of the commissural connections, bilateral synchronization of the rhythmic activities in the limbic system, commissural propagation of the epileptiform activities, etc. (+info)
A new sugar chain of the proteinase inhibitor from latex of Carica papaya.
The structure of a sugar chain of the proteinase inhibitor from the latex of Carica papaya was studied. Sugar chains liberated on hydrazinolysis were N-acetylated, and their reducing-end residues were tagged with 2-aminopyridine. One major sugar chain was detected on size-fractionation and reversed-phase HPLC analyses. The structure of the PA-sugar chain was determined by two-dimensional sugar mapping combined with sequential exoglycosidase digestion and partial acid hydrolysis, and by 750 MHz 1H-NMR spectroscopy. The structure found was Manalpha1-6(Manalpha1-3)Manalpha1-6(Manalpha1-3) (Xylbeta1-2)Manbeta1- 4GlcNAcbeta1-4(Fucalpha1-3)GlcNAc. This sugar chain represents a new plant-type sugar chain with five mannose residues. (+info)
2-Deoxyglucose selectively inhibits Fc and complement receptor-mediated phagocytosis in mouse peritoneal macrophages. I. Description of the inhibitory effect.
Incubation of normal or thioglycollate-elicited mouse peritoneal macrophages with 2-deoxy-D-glucose (2-dG) inhibits the capacity of these macrophages to phagocytize IgG- or complement-coated particles via their Fc and C3 receptors. 2-dG has no inhibitory effect on the capacity of these macrophages to phagocytize latex or zymosan particles, which are ingested in the absence of specific opsonins, and it does not inhibit binding of IgG- or C3-coated particles to their respective receptors on the macrophage's plasma membrane. 2-dG exerts its inhibitory effect on the macrophage and not on the opsonized particle. The inhibition is independent of particle size, occurs within 15-30 min of addition of this glucose analogue to the medium at 37 degrees C, cannot be overcome by supra-agglutinating amounts of opsonizing antibody, and is completely reversible by substitution of 5.5 mM glucose for 50 mM 2-dG in the medium. Addition of equimolar amounts of glucose or mannose, but not of fructose, galactose, fucose, or glucosamine, to medium containing 50 mM 2-dG results in substantial reversal of the inhibitory effect of 2-dG on Fc and C3 receptor mediated phagocytosis. (+info)
Discrimination of DNA hybridization using chemical force microscopy.
Atomic force microscopy (AFM) can be used to probe the mechanics of molecular recognition between surfaces. In the application known as "chemical force" microscopy (CFM), a chemically modified AFM tip probes a surface through chemical recognition. When modified with a biological ligand or receptor, the AFM tip can discriminate between its biological binding partner and other molecules on a heterogeneous substrate. The strength of the interaction between the modified tip and the substrate is governed by the molecular affinity. We have used CFM to probe the interactions between short segments of single-strand DNA (oligonucleotides). First, a latex microparticle was modified with the sequence 3'-CAGTTCTACGATGGCAAGTC and epoxied to a standard AFM cantilever. This DNA-modified probe was then used to scan substrates containing the complementary sequence 5'-GTCAAGATGCTACCGTTCAG. These substrates consisted of micron-scale, patterned arrays of one or more distinct oligonucleotides. A strong friction interaction was measured between the modified tip and both elements of surface-bound DNA. Complementary oligonucleotides exhibited a stronger friction than the noncomplementary sequences within the patterned array. The friction force correlated with the measured strength of adhesion (rupture force) for the tip- and array-bound oligonucleotides. This result is consistent with the formation of a greater number of hydrogen bonds for the complementary sequence, suggesting that the friction arises from a sequence-specific interaction (hybridization) of the tip and surface DNA. (+info)
Studies in calf venous pump function utilizing a two-valve experimental model.
OBJECTIVES: to explore the hydrodynamic mechanisms involved in the regulation of ambulatory venous pressure. DESIGN: an experimental model of calf venous pump was constructed with collapsible tubes and valves. MATERIAL: the model consisted of a conduit and a pump with an intervening competent valve. Another valve that could allow reflux into the pump was mounted above the pump. METHODS: conduit pressure and recovery times were monitored under conditions of different degrees of ejection fraction and reflux into the pump. Model variables included using poorly compliant tubes for the pump, the conduit and for both the pump and conduit. RESULTS: the latex tube exhibited a non-linear volume-pressure relationship and a bi-modal regimen of compliance. This bestowed pressure-buffering properties. Ambulatory venous hypertension resulted when reflux beyond buffering capacity occurred. Substituting less compliant PTFE for latex at the pump had a relatively minor effect on post-ejection pressure and recovery times. Using PTFE at the conduit had a profound but divergent effect on both of these parameters. Conduit capacitance reduction had a similar effect. CONCLUSION: conduit elastance plays a significant role in the regulation of ambulatory venous pressure in this experimental model. The hydrodynamic principles illustrated by the model may enhance our understanding of the human calf venous pump. (+info)
Separation of submicron bioparticles by dielectrophoresis.
Submicron particles such as latex spheres and viruses can be manipulated and characterized using dielectrophoresis. By the use of appropriate microelectrode arrays, particles can be trapped or moved between regions of high or low electric fields. The magnitude and direction of the dielectrophoretic force on the particle depends on its dielectric properties, so that a heterogeneous mixture of particles can be separated to produce a more homogeneous population. In this paper the controlled separation of submicron bioparticles is demonstrated. With electrode arrays fabricated using direct write electron beam lithography, it is shown that different types of submicron latex spheres can be spatially separated. The separation occurs as a result of differences in magnitude and/or direction of the dielectrophoretic force on different populations of particles. These differences arise mainly because the surface properties of submicron particles dominate their dielectrophoretic behavior. It is also demonstrated that tobacco mosaic virus and herpes simplex virus can be manipulated and spatially separated in a microelectrode array. (+info)
Adhesive properties of the isolated amino-terminal domain of platelet glycoprotein Ibalpha in a flow field.
We have examined the interaction between the amino-terminal domain of platelet glycoprotein (GP) Ibalpha and immobilized von Willebrand Factor (vWF) under flow conditions in the absence of other components of the GP Ib-IX-V complex. Latex beads were coated with a recombinant fragment containing GP Ibalpha residues 1-302, either with normal sequence or with the single G233V substitution that causes enhanced affinity for plasma vWF in platelet-type pseudo-von-Willebrand disease. Beads coated with native fragment adhered to vWF in a manner comparable to platelets, showing surface translocation that reflected the transient nature of the bonds formed. Thus, the GP Ibalpha extracellular domain is necessary and sufficient for interacting with vWF under high shear stress. Beads coated with the mutated fragment became tethered to vWF in greater number and had lower velocity of translocation than beads coated with the normal counterpart, suggesting that the G233V mutation lowers the rate of bond dissociation. Our findings define an approach for studying the biomechanical properties of the GP Ibalpha-vWF bond and suggest that this interaction is tightly regulated to allow rapid binding at sites of vascular injury, while permitting the concurrent presence of receptor and ligand in the circulation. (+info)