Enhanced activation of a T cell line specific for acetylcholine receptor (AChR) by using anti-AChR monoclonal antibodies plus receptors

Immune complex-mediated regulation of the immune response has been studied by using T cell lines and monoclonal antibodies (MAb), both specific for the acetylcholine receptor (AChR). Rat T lymphocytes bearing the W3/25 phenotype and specific for AChR from Torpedo californica have been propagated in vitro for nearly 1 yr. These T cells proliferate in response to optimal concentrations of AChR presented by irradiated syngeneic thymus cells. At suboptimal concentrations of antigen there is little activation of the T cell line. We report here that the addition of small amounts of anti-AChR MAb produces dramatic stimulation of the T cell lines at suboptimal doses of AChR. Enhanced activation depends on the isotype and not the fine specificity of the MAb that are used. The observed phenomenon is antigen specific, and in fact, the immune complexes may actually suppress the proliferative response of irrelevant T cells to some extent. The MAb plus antigen are rapidly bound to the surface of the antigen-presenting cell, which we have shown is the dendritic cell.     

Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor

Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the alpha and beta subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na[3H]BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, alpha and beta polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na[3H]BH4 in the presence of 8 M urea. The labeled alpha and beta polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the alpha and beta subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2', 4'-dinitrophenyl)-3-amino-propionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane.     

Ecology and community dynamics of Kubo people in the tropical lowlands of Papua New Guinea

Kubo producer-units (families and independent bachelors) could have been self-sufficient in the production of bananas but chose not to be. Nor did they seek self-sufficiency in the production of any combination of staple carbohydrate foods (bananas, tubers, sago flour) or, in the long term, strive for balance in the exchange of food with other producer-units. Despite the fact that bananas, which provided 50% of people's energy needs, were a delayed-return crop Kubo communities were very unstable. This instability and the failure to choose the option of self-sufficiency were connected and were mediated through intense intracommunity sharing that, ultimately, served to negotiate a concern with sorcery. The people grew bananas in the way they did, not out of environmental necessity, but to accommodate the crop to the needs of sharing and, thereby, facilitate community living.     

Thermal environment and sudden infant death syndrome: case-control study.

OBJECTIVE--To compare the thermal environment of infants who died of the sudden infant death syndrome with that of age matched control infants. DESIGN--Case-control study. Infants who died were matched with two controls, one for age and one for age and birth weight. Thermal measurements were conducted at the death scene for cases and at the scene of last sleep for control infants, who were visited unexpectedly within four weeks of the index infant''s death on a day of similar climatic conditions. A follow up questionnaire was administered to parents of cases and controls. SETTING--The geographical area served by the professional Tasmanian state ambulance service, which includes 94% of the Tasmanian population. SUBJECTS--41 infants died of the sudden infant death syndrome at home; thermal observations at death scene were available for 28 (68%), parental questionnaire data were available for 40 (96%). 38 controls matched for age and 41 matched for age and birth weight. RESULTS--Cases had more excess thermal insulation for their given room temperature (2.3 togs) than matched controls (0.6 togs) (p = 0.009). For every excess thermal insulation unit (tog) the relative risk of the sudden infant death syndrome was 1.26 (95% confidence interval 1.05 to 1.52). The average thermal bedding value calculated from parental recall was similar to that observed by attendant ambulance officers (mean difference = 0.4 tog, p = 0.39). Cases were more likely to have been found prone (odds ratio 4.58; 1.48 to 14.11). Prone sleeping position was not a confounder or effect modifier of the relation between excess thermal insulation and the syndrome. CONCLUSIONS--Overheating and the prone sleeping position are independently associated with an increased risk of the sudden infant death syndrome. Further work on infant thermal balance and sudden infant death is required and guidelines for appropriate infant thermal care need to be developed.     

Solubilization of native actin monomers from human erythrocyte membranes

Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I.     

Guanidine block of single channel currents activated by acetylcholine

The acetylcholine-activated channel of chick myotube was studied using the patch-clamp method. Single channel current amplitudes were measured between -300 and +250 mV in solutions containing the permeant ions Cs+ and guanidine (G+). G+ has a relative permeability, PG/PCs, of 1.6, but carries no more than half the current that Cs+ does, with an equivalent electrochemical driving force. Experiments using G+ revealed an asymmetry of the acetylcholine-activated channel, with G+ being more effective at reducing Cs+ currents when added to the outside than when added to the inside. The block caused by outside, but not inside, G+ was evident for both inward and outward currents. The block caused by outside G+ was voltage dependent, first increasing and then being partially relieved when the driving force was made more negative. Experiments with mixtures of Cs+ and G+ revealed anomalously low magnitudes for reversal potentials, relative to predictions based on the Goldman-Hodgkin-Katz equation. These findings are consistent with a two-well, three-barrier Eyring rate model for ion flow, and demonstrate that a highly permeant ion, guanidine, can block asymmetrically by acting from within the voltage field of the acetylcholine-activated channel.     

Secretion of metalloproteinases by stimulated capillary endothelial cells. I. Production of procollagenase and prostromelysin exceeds expression of proteolytic activity

We have characterized the biosynthesis of two metalloproteinases, procollagenase and prostromelysin, by rabbit brain capillary endothelial cells (RBCE) by means of immunochemical, biosynthetic, and functional assays. Unstimulated RBCE secreted no detectable metalloproteinases. Secretion of both procollagenase and prostromelysin was induced within 6 h by treating the cells with 50 ng/ml 12-O-tetradecanoylphorbol-13-acetate. In treated cells, the two proenzymes accounted for up to 20% of the [35S]methionine-labeled secreted proteins; about 15 micrograms of each protein was secreted in 48 h by 10(6) RBCE. Although RBCE secreted approximately as much procollagenase and prostromelysin as did rabbit fibroblasts, virtually no enzyme activity could be measured in RBCE-conditioned medium, even after activation of the proenzymes by trypsin or an organomercurial agent.     

Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.     

Recognition of Leishmania donovani antigens by murine T lymphocyte lines and clones. Species cross-reactivity, functional correlates of cell-mediated immunity, and antigen characterization

Studies in man and experimental animals suggest that cell-mediated immunity is of primary importance in limiting the pathogenesis of cutaneous and visceral leishmaniasis. In an attempt to determine, more directly, the role of T lymphocytes and the nature of the antigens that activate them, we have propagated antigen-specific murine T lymphocyte lines and clones that proliferate in response to antigens present on the membrane of intact Leishmania donovani promastigotes. One such line cross-reacts with membrane antigens on seven other Leishmania species and, to a lesser extent, with antigens on African procyclic trypanosomes. T lymphocyte clones that also exhibited a broad range of species cross-reactivity were isolated. About 40% of these clones had highly restricted specificity, whereas 60% were more extensively cross-reactive. The parent line and some clones passively transferred footpad DTH when injected locally, and some secreted a lymphokine activity that elicited intracellular killing of amastigotes within infected macrophages. Although the proliferative response of most clones was H-2 restricted, two clones appeared to be reactive in the presence of allogeneic antigen presenting cells. The majority of the clones appeared to recognize carbohydrate containing antigens, and absorption with solid substrate-bound lectins indicated that these antigens contained both mannose and galactose ligands. The antigenic activity was also absorbed using either of two extensively cross-reactive anti-parasite monoclonal antibodies.