Rachel Goldman

Enhancement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence.

Abstract The undecapeptide Substance P stimulates phagocytosis by mouse macrophages and human polymorphonuclear leukocytes. The activity of Substance P resides in its N-terminal tetrapeptide protion. Substance P and its N-terminal tetrapeptide are as active as tuftsin in their phagocytosis-stimulating activity and compete with tuftsin for its binding sites. The phagocytosis-enhancing activity of Substance P may play a role in inflammatory processes of neural origin where the involvement of the peptide has been implicated.

Kinetic behavior of a two-enzyme membrane carrying out a consecutive set of reactions.

Abstract A theoretical analysis of the kinetic behavior of a two-enzyme membrane carrying out two consecutive reactions is presented. The behavior of the immobilized enzyme system is compared to that of an analogous system consisting of the two enzymes in solution. The analysis of the immobilized enzyme system is based on the assumption that a quasi-stationary state is established within the unstirred layer at the membrane-solution interface. At this state, the rate of flow of the product formed by the first enzyme into the bulk of the solution, equals the difference between the rate of its production and the rate of its consumption by the second enzyme. The following boundary conditions were chosen in the kinetic analysis presented: (a) the activity of the first enzyme is independent of the second, (b) the enzymic reactions follow first order kinetics, (c) the concentration of the substrate of enzyme 1 is constant throughout the enzymic reaction, (d) the concentration of the products of both enzymic reactions are time dependent, (e) the system is of a finite volume. The effect of the kinetic parameters and of the physical characteristics of the system on its kinetic behavior are examined. It is shown that for all of the hypothetical systems that were analyzed the concentrations of the products of enzyme 1 and 2 in the bulk of the solution, P b 1 and P b 2 respectively, increase linearly with time at least for the first 10 minutes of reaction. For systems which consist of enzymes of relatively high activity, the enzymic reactions are diffusion controlled; the activity of enzyme 1 is limited by the rate of diffusion of substrate from the bulk of the solution, and the activity of enzyme 2 approaches that exhibited by enzyme 1. An analysis of a corresponding homogeneous system containing both enzymes in solution reveals the appearance of an initial lag period in the production of P b 2 . The length of the lag period is determined by the catalytic as well as the physical parameters of the system. In the systems studied, the concentration of P b 1 approaches with time a limiting value. The higher the activity of the two enzymes the sooner the stationary state with respect to P b 1 is reached, and the value of P b 1 is lower. The rate of production of the end product at the first stages of the reaction is markedly higher in the immobilized enzyme system than that predicted for a corresponding homogeneous system.

Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi

The attachment of Escherichia coli and Salmonella typhi to mouse peritoneal macrophages was inhibited by D-mannose, methyl α-D-mannopyranoside and yeast mannan, but not by any other sugar tested. D-Mannose and its derivatives also inhibited the attachment of E. coli to human polymorphonuclear leucocytes. Mannan inhibited phagocytosis when preincubated with E. coli, but not when preincubated with leucocytes. Attachment of opsonized bacteria to leucocytes was not inhibited by D-mannose or methyl α-D-mannopyranoside nor by any other sugar tested. Our results suggest that the surface of phagocytes, like that of epithelial cells, contains D-mannose residues which serve for the attachment of certain Gram negative bacteria.

A differential interaction of daunomycin, adriamycin and their derivatives with human erythrocytes and phospholipid bilayers.

Drug-membrane association of daunomycin, adriamycin and three of its derivatives, adriamycin-14-octanoate (AD-14-OCTA), adriamycin-14-acetate (AD-14-ACE) and N-trifluoroacetyladriamycin-14-valerate (AD32), was studied using phospholipid bilayers and human erythrocytes. The various drugs exhibited a differential affinity to membrane-lipid domains. Lipid-incorporated drugs exhibit a marked change in the shape of the emission spectrum which was utilized for the evaluation of the apparent dielectric constant, epsilon, of the environment surrounding the anthracycline moiety, as well as for the determination ofthe partitioning constant. By measuring the fluorescence polarization and the fluorescence lifetime of the incorporated drugs, rotational relaxation times of 4--8 ns were derived. These parameters provide a supportive evidence of the association of the fluorophore of the drugs with membrane-lipid domains. The anthracycline derivatives interact to a different degree with dipalmitoyl phosphatidylcholine and phosphatidylserine as reflected by changes in their thermotropic properties assessed by differential scanning calorimetry. Daunomycin was the most effective in decreasing the temperature of the phase transition and brought about a comparable reduction in the enthalpy of melting as AD32 and AD-14-OCTA. Adariamycin was the least potent of the series. AD-14-ACE and AD32 protected erythrocytes against hypotonic lysis, adriamycin and daunomycin had no significant effect on the susceptibility to hypotonic lysis, whereas AD-14-OCTA proved to be hemolytic even at low concentration (approx. 10(-7M).

Characteristics of the β-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.

Rupture of rat liver lysosomes mediated by L-amino acid esters.

Abstract Treatment of rat liver lysosome suspensions with 0.5–20 mM α- l -amino acid esters results in a progressive loss of latency of lysosomal enzyme activity. The increase in available acid phosphatase activity in lysosomal suspensions is correlated with the decrease of turbidity of these suspensions. Ester mediated turbidity decrease is dependent upon ester concentration, and the pH and ionic strength of the suspending medium. d -Stereoisomers of amino acid esters do not exhibit comparable capacity to damage lysosomes. α- l -Amino acid esters were found to be substrates for neutral lysosomal esterase and transpeptidase activity. The d -stereoisomers are degraded at much lower rates. These data support the hypothesis that ester dependent lysosomal rupture is mediated by the specific interaction of the ester with a structural or functional lysosomal protein.

Reactive oxygen species are involved in the activation of cellular phospholipase A2.

Vanadate (V) potentiated (4‐ to 10‐fold) the activation of cellular phospholipase A2 (PLA2) induced by H2O2 (H), a phorbol ester (T), a Ca2+‐ionophore (A) and opsonized zymosan in macrophages. V+H induced in intact cells the activation and translocation of PLA2 and protein kinase C(PKC) to the plasma membrane. V+H and V+T+A induced strong chemiluminescence (CL) which was abrogated by a specific NADPH oxidase inhibitor diphenylene iodonium (DPI). DPI markedly suppressed the stimulation of PLA2 by V+T+A and V+OZ. The results suggest that the formation of endogenous reactive oxygen species (ROS) is important for PLA2 activation.

ION DISTRIBUTION IN LYSOSOMAL SUSPENSIONS

Lysosomes are vesicular subcellular organelles containing a variety of hydrolytic enzymes with a characteristic acid pH optima. It has been inferred that the pH within the lysosomes is low although no direct evidence is available to support this claim [ 11. Various basic dyes and other cationic molecules have been shown’to be concentrated within lysosomes [2]. Amine distribution has been used to determine the internal pH in subcellular organelles in which the internal pH is lower than that of the medium [3]. We studied the distribution of amines and other cations in a lysosomal suspension in order to find whether there is a ApH and if there is what is its origin. Despite their apparent permeability to salts lysosomes demonstrate remarkable osmotic stability in salt solution [4]. Our results demonstrate the existence of various ion gradients, including a proton gradient, which are due to Donnan distribution, and explain the osmotic stability of lysosomes on the basis of a Donnan dependent salt exclusion.

On the mechanism of action of the phagocytosis-stimulating peptide tuftsin.

SummaryIncubation of human polymorphonuclear leukocytes (PMNL) or thioglycollate-stimulated mouse peritoneal macrophages with the phagocytosis-stimulating peptide, tuftsin (2.5 × 10−7 M, at 37 °C), caused an increase of 89–90% in intracellular cGMP levels, accompanied by a decrease of 20–25% in intracellular cAMP levels. Significant changes in cyclic nucleotide levels were detectable after 4 min of incubation, were maximal at 10–20 min and persisted for at least 60 min. The concentration dependences of the stimulatory effect of tuftsin on modulation of intracellular levels of cyclic nucleotides and on phagocytosis are similar, suggesting a cause and effect relationship between the two phenomena. This notion is further supported by the finding that 8-Br-cGMP and 8-Br-cAMP elicit stimulatory and inhibitory effects on macrophage phagocytosis, respectively. Measurement of 45Ca2+ influx into PMNL and macrophages in the presence and absence of tuftsin did not reveal any change in 45Ca2+ uptake from the media. However, tuftsin did enhance release of 45Ca2+ from cells preloaded with the isotope. Results suggest that modulation of both the amount of cell-associated 45Ca2+ and the intracellular levels of cyclic nucleotides are key steps in the mechanism by which tuftsin augments phagocytosis.

Effect of cannabinoids on neurotransmitter uptake, atpase activity and morphology of mouse brain synaptosomes.

Δ1-Tetrahydrocannabinol (Δ1-THC) and cannabidiol (CBD), a psychoactive and a nonpsychoactive constituent of marijuana respectively, inhibit the uptake of 3H-labelled norepinephrine (NE), dopamine (DA), γ-aminobutyric acid (GABA) and serotonin (5-HT), by mouse brain synaptosomes. CBD is more effective than Δ1-THC in the inhibition of neurotransmitter uptake. At 5 × 10−5 M both CBD and Δ1-THC inhibit uptake by 60–100%. The one exception to the above is the differential effect of Δ1-THC and CBD on 5-HT uptake. At 10−6 M of Δ1-THC the uptake is twice that of the control value and at 5 × 10−5 M uptake is still equal to control value. At the former concentration CBD has no effect on 5-HT uptake whereas at the latter concentration a 50 per cent inhibition is observed. Both Δ1-THC and CBD inhibit Na+-K+-ATPase and Mg-ATPase activities; at 5 × 10−5 M inhibition amounts to 40 per cent. Electron microscopy reveals that synaptosomal preparations are highly damaged at 5 × 10−5 M. Thus inhibition of uptake could stem from either failure of ATPase activity, from disruption of synaptosomes, or from both.