Induction by mycoplasmas of tumor necrosis factor-alpha from macrophages

Several species of mycoplasmas including M. pneumoniae, the causative agent of human respiratory infection, were investigated for tumor necrosis factor-alpha (TNF-alpha) induction. The cytotoxic activity to Meth A cells of peritoneal macrophages purified from BALB/c mice was enhanced markedly when cultured with either viable or nonviable mycoplasmas. The supernatant of macrophage culture mixed with mycoplasmas, M. pneumoniae or A. laidlawii, showed a potent cytotoxic activity to TNF-alpha-sensitive but not to TNA-alpha-insensitive L cells. Addition of anti-TNA-alpha antiserum inhibited completely the cytotoxic activity of the supernatant, indicating that the cytotoxic activity is due mostly to TNF-alpha. These results strongly suggest that mycoplasmas possess an activity to induce TNF-alpha, which enhances the cytotoxic activity of macrophages and prevent infection with mycoplasmas in vivo.     

Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation

The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5'-monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Differential adaptation of membranes of two osmotolerant fungi,Aspergillus chevalieri and Penicillium expansum to high sucrose concentrations

Aspergillus chevalieri and Penicillium expansum were able to tolerate sucrose concentrations in the growth media up to 80% (w/v). At 50% sucrose the growth rate is approximately 1.4 and 1.2 times, respectively, higher than in the control. While at 80% sucrose it drops to 35% and 45% of the control level for both fungi. Lipids and proteins in plasma membranes increased with increasing sucrose concentrations in the growth medium. Phospholipid content in membranes of both organisms being also increased, phosphatidyl glycerol was the major detected phospholipid and represented the highest increase. The fatty acid composition of fraction enriched plasma membrane of both fungi changed when they were grown in high sucrose concentrations. Some fatty acids which had not been detected in control cultures were present and the proportions of other fatty acids changed. At 50% sucrose the unsaturation index of membranes decreased by 20-25% in both fungi, indicating that the plasma membrane is less fluid at this concentration. At 80% sucrose a similar trend was observed for P. expansum but for A. chevalieri the unsaturation index was little changed compared with the control. The fluorescence polarization values of 1,6-diphenyl 1,3,5-hexatriene (DPH) in membranes of both fungi grown at 80% sucrose increased, indicating a decrease in membrane fluidity. At 50% sucrose the increase in saturation of membrane fatty acids would tend to reduce membrane fluidity but in A. chevalieri at 80% sucrose fatty acids did not become more saturated. In this case the marked increase in sterols at this sucrose concentration may be responsible for low membrane fluidity.     

Cytolytic activity of Naegleria fowleri cell-free extract

The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30-60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by 51Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50 degrees C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing 51Cr release from target cells by the soluble fraction of N. fowleri extracts.     

A vacuolar-type ATPase, partially purified from potassium transporting plasma membranes of tobacco hornworm midgut

An azide- and vanadate-insensitive, N-ethylmaleimide-sensitive ATPase has been partially purified from a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. The properties of the membrane-bound ATPase activity were identical to those of the ATPase activity of highly purified goblet cell apical membranes (Wieczorek, H., Wolfersberger, M. G., Cioffi, M., and Harvey, W. R. (1986) Biochim. Biophys. Acta 857, 271-281). 90% of the azide- and vanadate-insensitive ATPase activity was solubilized by C12E10, leaving 90% of the contaminating azide-sensitive mitochondrial ATPase activity in the pellet after centrifugation at 100,000 x g for 1 h. After discontinuous sucrose gradient centrifugation of the supernatant at 220,000 x g for 1 h nearly all of the azide- and vanadate-insensitive ATPase activity was found in the 30% sucrose fraction without contaminating azide- or vanadate-sensitive ATPase activity. Two prominent bands with relative molecular masses (Mr) of about 600,000 and 900,000, both displaying azide-insensitive and N-ethylmaleimide-sensitive ATPase activity, were found in native microgradient polyacrylamide gel electrophoresis of the 30% sucrose fraction. The two bands could not be separated by anion exchange chromatography. Denaturation of both bands resulted in the same polypeptide pattern (five major bands with Mr 70,000, 57,000, 46,000, 29,000 and 17,000) in sodium dodecylsulfate-polyacrylamide gel electrophoresis, indicating that they represented oligomers of the same protein unit. Substrate and inhibitor specificities of the partially purified ATPase were similar to those of the membrane-bound ATPase activity, whereas salt selectivity differed partly. Altogether, structural and functional properties of the ATPase strongly resemble those of vacuolar-type ATPases.     

Cytolytic Activity of Naegleria fowleri Cell-free Extract1

ABSTRACT. The cytotoxic activity of a cell-free extract of Naegleria fowleri amebae on B103 rat nerve cells in culture was investigated. The cell-free extract was prepared by subjecting lysed amebae to centrifugation at 100,000 g for 1 h, precipitation of the supernatant fluid with 30–60% saturated ammonium sulfate, and desalting by group exclusion chromatography utilizing Sephadex G-25. The supernatant fluid recovered from this procedure was termed the soluble fraction. The Naegleria cytotoxic activity present in the soluble fraction was assayed by ⁵¹Cr released from labeled B103 cells. The Naegleria soluble fraction, when added to nerve cells, elicited blebs on the B103 target cell surface within 5 min after exposure to the fraction. Later, holes were observed in the B103 cell plasma membrane. These alterations were never observed on untreated B103 cells. Phospholipase A, phospholipase C, and protease activities were associated with the desalted ammonium sulfate-precipitable cytotoxic activity of N. fowleri cell-free lysate. The cytotoxic activity was impaired by ethylenediamine-tetraacetate (EDTA), phospholipase A inhibitor (Rosenthal's reagent), heating at 50°C for 15 min, or incubation at pH 10 for 60 min. Repeated freeze-thawing and inhibitors of proteolytic enzymes had no effect on the cytotoxic activity. Small amounts of ethanol (5% v/v) enhanced cytotoxic activity of the fraction. Phospholipases A and C, as well as other as yet unidentified cytolytic factors may be responsible for producing ⁵¹Cr release from target cells by the soluble fraction of N. fowleri extracts.     

PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen     

Release of adenylate cyclase stimulating, GTP-binding protein, Gs from human erythrocyte membranes under the effect of fluoride-ion and guanine nucleotides

Human erythrocyte membranes were incubated in the presence of sodium fluoride or guanylylimidodiphosphate (GppNHp), a nonhydrolysable GTP analog. After centrifugation at 100000 g the activity of the adenylate cyclase-stimulating GTP-binding protein, Gs, was detected in the supernatant fraction. The release of the Gs activity from the membranes closely resembles Gs activation by GppNHp. The Gs activity release from the GppNHp-induced membranes is characterized by a lag period. The nucleotide concentration causing a half-maximal solubilization is about 9.10(-7) M. Approximately 50% of the Gs activity released from sodium fluoride-treated human erythrocyte membranes was associated with the cytoskeletal fraction extracted by a low ionic strength solution. The data obtained suggest that Gs exists in the membrane at lease in two compartmentalized states and is solubilized from both states during its activation.     

Properties of 5'-nucleotidase in rat heart sarcolemma

The activity of 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) was examined in membrane fractions isolated by hypotonic shock-LiBr treatment (fraction HL) and sucrose gradient separation (fraction S) of rat ventricle homogenate. The enzyme activity in these two fractions differed significantly in several respects. In fraction HL, 5'-nucleotidase had a high affinity for AMP (Km 35 microM), and ATP was a potent competitive inhibitor. In contrast, the 5'-nucleotidase displayed by fraction S showed a low substrate affinity (Km 130 microM) and less sensitivity to ATP. Treatment of membranes with trypsin and neuraminidase markedly stimulated 5'-nucleotidase in fraction HL, whereas only a modest effect was observed in fraction S. Exposure of the membranes to Triton X-100 resulted in a 60% and 10% increase in the enzyme activity in fractions HL and S, respectively. The characteristic activity ratios of 5'-nucleotidase at 200 microM relative to 50 microM AMP in fractions HL and S were modified by alamethicin in an opposite way and became identical. Although concanavalin A almost completely inhibited the 5'-nucleotidase activity in both membrane preparations at a concentration of 2 microM, Hill plots of the data on concanavalin A inhibition revealed a coefficient of 2.2 for fraction S and 1.1 for fraction HL. The differences in 5'-nucleotidase activity of the two membrane fractions are considered to be due to differences in the orientation of the vesicles of the sarcolemmal preparations. These results suggest that two distinct catalytic sites for 5'-nucleotidase are present at the intra- and extracellular surface of the rat heart sarcolemma.     

Persistence of Staphylococcus aureus L-form during experimental lung infection in rats

The course of pulmonary infection in rats infected by intranasal inoculation with a Staphylococcus aureus stable protoplast L-form was studied. Blood and bronchoalveolar samples were taken on days 3, 7, 14 and 30 after challenge and were investigated by microbiological, electron-microscopic, cytochemical and cytometric methods. The electron microscopic data and isolation of L-form cultures from bronchoalveolar samples at all experimental times demonstrated the ability of S. aureus L-form cells to internalize, replicate and persist in the lungs of infected rats to the end of the observation period, in contrast to the S. aureus parental form. It was found that persisting L-form evoked ineffectual phagocytose by alveolar macrophages and low but long-lasting inflammatory reaction in rats. The experimental model of pulmonary infection with S. aureus L-form suggests that the cell-wall-deficient bacterial forms may be involved in the pathogenesis of chronic and latent lung infections.     

Interactions of Cations with Membrane Fractions of Smooth and Rough Strains of Salmonella typhimurium and Other Gram-Negative Bacteria

Addition of cations (20 to 50 mM for Mg²⁺ or Ca²⁺ or 100 to 500 mM for Na⁺) to N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer during preparation of membranes from smooth and rough strains of Salmonella typhimurium LT2, Salmonella minnesota, and Escherichia coli O8 had two effects on the composition of the membranes isolated. First, in rough strains of chemotypes Ra to Re the “total membranes” (pellets from high-speed centrifugation) were deficient in the proteins of the outer membrane. The missing proteins were found to have been sedimented in a prior low-speed centrifugation in a fraction we call “cation-aggregated membranes.” Since these membranes were enriched for lipopolysaccharide and for outer membrane proteins, deficient in succinic dehydrogenase, and contained primarily the dense peak after sucrose gradient centrifugation, it appears to be relatively pure outer membrane. About 10% of the membrane protein of smooth strains and up to 50% that of rough strains were cation-aggregated membranes, appearing to contain most of the outer membrane of rough strains. Thus, cation aggregation may be a useful means of preparation of outer membrane samples. The second effect was that with cation addition, several high-molecular-weight proteins not seen when membranes were prepared without cation addition were found in the total membranes of both smooth and rough strains after high-speed centrifugation. These proteins were bound by cations to the inner membranes, since they were soluble in Triton X-100 and separated into the less dense peak upon sucrose gradient centrifugation. They originated from the cytoplasm or the periplasm, since they corresponded to soluble proteins found in the supernatant after high-speed centrifugation and were depleted from this supernatant when preparation was done in the presence of cations.     

Staphylococcal alpha-toxin synergistically enhances inflammation caused by bacterial components

This study was performed to investigate the in vivo effects of staphylococcal alpha-toxin on phagocytosis and the secretion of proinflammatory cytokines at local sites of intraperitoneal toxin-challenged mice. A dosage of 45 hemolytic units (HU) of alpha-toxin induced a marked increase in the peritoneal neutrophil count. The toxin caused a 52% decrease in phagocytosis by peritoneal macrophages, compared with that of control mice receiving Staphylococcus aureus particles alone. However, no effect on phagocytosis in neutrophils was observed. A dosage of 45 HU toxin and the synergistic activity of S. aureus particles strongly induced interleukin (IL) 6 secretion but only mildly induced IL-1alpha secretion. The toxin did not induce the secretion of tumor necrosis factor-alpha (TNF-alpha). Interestingly, S. aureus culture supernatant induced the secretion of TNF-alpha in cultured macrophages. These results suggest that alpha-toxin damages the primary host defense system by inducing the oversecretion of IL-1alpha and IL-6, but not TNF-alpha, via a mechanism that requires the synergistic action of bacterial components.     

Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Fatty Acid Composition of L-Forms of Streptococcus faecalis Cultured at Different Osmolalities

The fatty acid composition of the membranes of three different penicillin-produced L-forms of Streptococcus faecalis was determined: (i) a stable (nonreverting) L-form (T(53)) cultured in brain heart infusion (BHI) with 0.5 M sucrose; (ii) a stable L-form (T(531)) cultured in BHI without sucrose; and (iii) an unstable L-form (T(9)) cultured in BHI with 0.5 M sucrose and 1,000 U of penicillin per ml. L-forms were obtained by centrifugation and lysed by washing in 1 mM tris(hydroxymethyl)aminomethane-hydrochloride buffer. The parent S. faecalis was also cultured in BHI and BHI containing 0.5 M sucrose, and washed with buffer. The fatty acid composition of L-forms of S. faecalis cultured in BHI without sucrose (370 mosmol) had higher C(18:1) and lower C(18) than L-forms cultured in the same media with added 0.5 M sucrose (950 mosmol) in both exponential and stationary cultures. In the stationary phase of growth, C(19) was reduced in the L-forms cultured without sucrose. Similar changes were seen in the parent S. faecalis cultured in the two types of media. These changes in membrane fatty acids may relate to osmo-regulation of the L-forms.     

Water-soluble prolactin receptors from porcine mammary gland

Two types of prolactin receptors were identified in sow mammary gland. When light membranes were prepared on a discontinuous sucrose gradient (0.3 and 1.7 M) and then diluted and washed with 0.3 M sucrose solution, a large amount (about 50%) of receptors were released from membranes and appeared in the supernatant fraction. These two forms (hydrophobic and water-soluble) of receptors were characterized as having the same binding specificity for lactogenic hormones and a similar affinity constant for ovine prolactin (K alpha approximately 10-12 X 10(9) M-1). Polyclonal antibodies and one monoclonal (mAb M110) antibody, obtained against partially purified prolactin receptors from rabbit mammary gland, cross-reacted effectively with sow mammary receptors. They completely inhibited the specific binding of [125I]oPRL to membrane and water-soluble receptors. The present studies indicate that the two types of sow prolactin receptors could represent the same molecular entity and confirm that prolactin receptors from rabbit and sow mammary gland exhibit numerous antigenic similarities.     

The aging of a brain soluble fraction modifies its effect on the activity of neuronal Na+, K+-ATPase

In previous work we presented evidence showing that a brain soluble fraction was necessary to observe the stimulation of membrane Na+,K+-ATPase activity by catecholamines. Preliminary experiments suggested to us that the soluble fraction by itself was able to modify this enzyme activity. In the present study we have assayed the activity of synaptosomal Na+,K+-ATPase in the presence of a soluble fraction (aqueous supernatant after 100,000 g 30 min) prepared from rat cerebral cortex. The soluble fraction was used at different times after its preparation and different conditions in the incubation period previous to the enzyme assay were tested. It was observed that the enzyme activity increased 70% in the presence of a "0 min" soluble fraction. This effect was not found: a) in the presence of a "30 min" soluble fraction or b) when the membranes plus a "0 min" soluble fraction were incubated for 30 min (15 min at 37 degrees C + 15 min at 0 degree C) before the ATPase assay. In the presence of a "60 min" or "24 h" soluble fraction Na+,K+-ATPase activity was inhibited 50%. Results obtained indicate that Na+,K+-ATPase activity of synaptosomal membranes can be stimulated, inhibited or unchanged, depending on the aging of the soluble fraction.