The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils

Formyl-met-leu-phe (fMLP) induces actin assembly in neutrophils; the resultant increase in F-actin content correlates with an increase in the rate of cellular locomotion at fMLP concentrations less than or equal to 10(-8) M (Howard, T.H., and W.H. Meyer, 1984, J. Cell Biol., 98:1265-1271). We studied the time course of change in F-actin content, F-actin distribution, and cell shape after fMLP stimulation. F-actin content was quantified by fluorescence activated cell sorter analysis of nitrobenzoxadiazole-phallacidin-stained cells (Howard, T.H., 1982, J. Cell Biol., 95(2, Pt. 2:327a). F-actin distribution and cell shape were determined by analysis of fluorescence photomicrographs of nitrobenzoxadiazole-phallacidin-stained cells. After fMLP stimulation at 25 degrees C, there is a rapid actin polymerization that is maximal (up to 2.0 times the control level) at 45 s; subsequently, the F-actin depolymerizes to an intermediate F-actin content 5-10 min after stimulation. The depolymerization of F-actin reflects a true decrease in F-actin content since the quantity of probe extractable from cells also decreases between 45 s and 10 min. The rate of actin polymerization (3.8 +/- 0.3-4.4 +/- 0.6% increase in F-actin/s) is the same for 10(-10) - 10(-6) M fMLP and the polymerization is inhibited by cytochalasin D. The initial rate of F-actin depolymerization (6.0 +/- 1.0-30 +/- 5% decrease in F-actin/min) is inversely proportional to fMLP dose. The F-actin content of stimulated cells at 45 s and 10 min is greater than control levels and varies directly with fMLP dose. F-actin distribution and cell shape also vary as a function of time after stimulation. 45 s after stimulation the cells are rounded and F-actin is diffusely distributed; 10 min after stimulation the cell is polarized and F-actin is focally distributed. These results indicate that actin polymerization and depolymerization follow fMLP stimulation in sequence, the rate of depolymerization and the maximum and steady state F-actin content but not the rate of polymerization are fMLP dose dependent, and concurrent with F-actin depolymerization, F-actin is redistributed and the cell changes shape.     

Bulinus tropicus, a natural intermediate host for Schistosoma margrebowiei in Lochinvar National Park, Zambia

Snails, provisionally identified as Bulinus tropicus, on the basis of chromosome number, egg protein profile, AcP and HBDH enzymes of the digestive gland, and radular morphology, from Lochinvar National Park, Zambia have been demonstrated to transmit Schistosoma margrebowiei naturally. The identification of the unpaired male schistosomes was confirmed by PGM and AcP analyses. The observations confirm earlier epidemiological predictions, and add another species of mollusc to the two, B. forskalii and B. scalaris, known to be natural intermediate hosts of S. margrebowiei.     

Prophylactic immunization against experimental leishmaniasis. IV. Subcutaneous immunization prevents the induction of protective immunity against fatal Leishmania major infection

Durable immunity against fatal L. major infection in genetically susceptible mice can be induced by immunization with 150,000-rad irradiated or heat-killed promastigotes administered i.v. or to a lesser extent i.p. Conversely, subcutaneous (s.c.) and intramuscular (i.m.) injections are not only totally ineffective but generally increase susceptibility to and enhance the progression of the disease, leading to earlier mortality. This detrimental effect is particularly evident with lower infecting challenge doses. Disease exacerbation is apparent in mice given 4 X s.c. injections of as few as 2 X 10(4) irradiated promastigotes, but it appears most potent after doses of 2 X 10(7). When mice given 4 X s.c. injections were subsequently immunized i.v. with 2 X 10(7) irradiated promastigotes, they failed to develop any evidence of protection against infection with 2 X 10(5) promastigotes, whereas mice given i.v. immunization alone were strongly protected. Thus, s.c. injections are capable of blocking the prophylactic effect of i.v. immunization with irradiated parasites. This inhibitory effect can be achieved with a single s.c. injection, although rather less potently than with four, and is even effective against four repeated weekly i.v. immunizations. Once induced, the effect persists undiminished after 100 days. A weaker effect is also inducible by s.c. injection given after i.v. immunization. The blocking effect of s.c. injection is not dependent on continuing viability of the promastigotes, as it can be induced equally readily with heat-killed, formalin-fixed, or sonicated parasites. The phenomenon extends to mouse strains genetically resistant as well as susceptible to L. major infection and, in congenic mice of BALB background, is independent of the major histocompatibility (H-2) gene complex.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Osmotic water permeability of small intestinal brush-border membranes

Summary A stopped-flow nephelometric technique was used to examine osmotic water flow across small intestinal brush-border membranes. Brush-border membrane vesicles (BBMV) were prepared from rat small intestine by calcium precipitation. Scattered 500 nm light intensity at 90° to incident was a linear function of the number of vesicles in suspension, and of the reciprocal of the suspending medium osmolality. When BBMV were mixed with hyperosmotic mannitol solutions there was a rapid increase in the intensity of scattered light that could be fit to a single exponential function. The rate constant for vesicle shrinking varied with temperature and the size of the imposed osmotic gradient. At 25°C and an initial osmotic gradient of 50 mOsm, the rate constant was 1.43±0.044 sec^–1. An Arrhenius plot of the temperature dependence of vesicle shrinking showed a break at about 25°C with an activation energy of 9.75±1.04 kcal/mole from 11 to 25°C and 17.2±0.55 kcal/mole from 25 to 37°C. The pore-forming antibiotic gramicidin increased the rate of osmotically driven water efflux and decreased the activation energy of the process to 4.51±0.25 kcal/mole. Gramicidin also increased the sodium permeability of these membranes as measured by the rate of vesicle reswelling in hyperosmotic NaSCN medium. Gramicidin had no effect on mannitol permeability. Assuming spherical vesicles of 0.1 m radius, an osmotic permeability coefficient of 1.2×10^–3 cm/sec can be estimated for the native brush-border membranes at 25°C. These fesults are consistent with the solubility-diffusion model for water flow across small intestinal BBMV but are inconsistent with the existence there of large aqueous pores.     

Electrogenic proton translocation by the ATPase of sugarcane vacuoles

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.     

Evidence for a plasmalemma redox system in sugarcane

A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O₂ consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K⁺, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH.     

Phospholipids and lipopolysaccharide of Aeromonas hydrophila.

The phospholipids and lipopolysaccharide of Aeromonas hydrophila were characterized. Phosphatidylethanolamine and phosphatidylglycerol were the major phospholipid components. The outer membrane contained more phosphatidylethanolamine and less phosphatidylglycerol than the inner membrane, and the phospholipids of the outer membrane contained a higher proportion of saturated fatty acids. Only four fatty acids (C14:0, C16:0, C16:1, and C18:1) were found in the phospholipids. The lipopolysaccharide of A. hydrophila did not contain the eight-carbon sugar 3-deoxyoctulosonic acid nor did it contain C16:0, both of which are typical constituents of the lipopolysaccharide of many other species.     

Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.