Lipid-dependent recovery of alpha-bungarotoxin and monoclonal antibody binding to the purified alpha-subunit from Torpedo marmorata acetylcholine receptor. Enhancement by noncompetitive channel blockers

Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.     

Isoelectric point determination of human and camel beta-endorphin, alpha-endorphin and enkephalins

The isoelectric point of the camel and the human β-endorphin, of the α-endorphin and the enkephalins were determined by analytical isoelectric focusing on 1 mm thin polyacrylamide gel slab. The difficulty of staining peptides as short as β-endorphin or smaller was overcomed using a modification of Bibring and Baxandall's or Faupel and Von Arx's staining method. The camel β-endorphin gives two bands having isoelectric point of 10.3 and 10.4, the human β-endorphin focus at pH 9.9, while α-endorphin, leu and met-enkephalin at pH 5.9, 5.5 and 5.45 respectively. The staining method described coupled with the isoelectric focusing seems to be fit for discriminating β-endorphin in a crude rat pituitary extract.     

Ultrastructure of the developing pigment strand of rice (Oryza sativa L.) in relation to its role in solute transport

Summary The developing pigment strand of rice (Oryza sativa L.) was studied by conventional electron microscopy and also by use of thick sections post-fixed with zinc iodide and osmium (ZIO).When the rice caryopsis achieves maximum length, a suberised adcrusting wall layer is laid down over the original primary walls of the pigment strand. Concomitant with suberin deposition a proliferation of tubular endoplasmic reticulum occurs in the cytoplasm giving rise to numerous interconnected vesicles which bear ribosomes. The vesicles in the general cytoplasm retain their ribosomes while those close to the wall become smooth and contain an electron-opaque granular material which is eventually deposited to the outside of the plasmalemma. This granular material may be the precursor(s) from which suberin is polymerised. The suberised wall attains about six times the width of the original primary wall and plasmodesmata, which traverse both primary wall and suberised wall layers, become greatly elongated.Lipid bodies increase in both size and frequency during development, eventually coalescing to form a complete plug across the pigment strand and occluding the symplast of this tissue. The significance of these ultrastructural observations is discussed in relation to the previously demonstrated role of the pigment strand as a translocation pathway for water and assimilates during grain filling.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium fixation     

Metabolism of palmitate in cultured rat Sertoli cells

Isolated rat Sertoli cells were incubated in the presence of [1-14C]palmitate at a cell concentration of 1.54 +/- 0.31 mg protein/flask (n = 7). The oxidation of palmitate was concentration dependent and maximal oxidation was obtained at 0.35 mM-palmitate. At a saturating concentration of palmitate the oxidation was linear for at least 6 h. About 65% of the total amount of palmitate oxidized during 5 h at 0.52 mM-palmitate (109 +/- 44 nmol/flask, n = 5) was recovered as CO2 and the rest as acid-soluble compounds. Almost all radioactive acid-soluble compounds which were secreted by the Sertoli cells were shown to be 3-hydroxybutyrate and acetoacetate. The palmitate recovery in cellular lipids and triacylglycerols was 9.4 +/- 5.1 nmol/flask (n = 5) and 3.5 +/- 2.8 nmol/flask (n = 5) respectively. Addition of glucose had no significant effect on palmitate oxidation but caused a 9-fold increase in esterification of palmitate into triacylglycerols. We conclude that cultured rat Sertoli cells can oxidize palmitate to CO2 and ketone bodies and that fatty acids appear to be a major energy substrate for these cells.     

Pulmonary clearance of inhaled 99mTc-DTPA: effects of surfactant depletion by lung lavage

The influence of surfactant depletion on clearance from the lungs of inhaled technetium-99m-labeled diethylenetriamine pentaacetate (99mTc-DTPA) was studied in rabbits. Surfactant was removed by repeated lung lavage with isotone saline. To minimize structural damage to the lungs, pressure generated insufflation with short expiration was utilized. Aerosolized 99mTc-DTPA was administered via a bag-in-bottle system. Radioactivity was measured with a gamma camera and time-activity curves were obtained over the base of the right lung. Six nonlavaged rabbits served as controls. In six lavaged rabbits clearance of 99mTc-DTPA was significantly faster than in controls. In three rabbits given natural surfactant into the trachea after lung lavage, 99mTc-DTPA was eliminated faster than in controls but slower than in surfactant-depleted animals. The results indicate a role of surfactant on clearance rate of 99mTc-DTPA from rabbit lungs. Measurements of 99mTc-DTPA clearance may be useful in studying the function of the surfactant system in different lung disorders.     

N.m.r. studies of metabolism in perfused organs

Several metabolites and intracellular pH in intact organs can be studied in a non-destructive manner by phorphorus nuclear magnetic resonance (31P n.m.r.). This possibility was demonstrated by us nearly five years ago. Since then we have developed the appropriate physiological techniques and improved the n.m.r. method for the study of animal hearts and kidneys. Here we described measurements aimed at clarifying three problesm. (1) Having measured the enzyme-catalysed fluxes between phosphocreatine and ATP by the method of saturation transfer n.m.r., we examine the relations between energy supply and heart rate in the isolated perfused rat heart. (2) We describe experiments to establish the validity of the perfusion model. For the first time, we report 31P n.m.r. measurements of an in vivo rat heart and compare the results with those obtained for the perfused rat heart. (3) Ischaemia and metabolism in rabbit kidneys is investigated to establish the relation between functional and metabolic recovery after a renal transplant operation.     

Sexual advertisement and immune function in an arachnid species (Lycosidae)

A simple version of the immunocompetence handicap hypothesizesthat through condition-dependence, the size of the sexual traitmay be positively related to immune function at the populationlevel. In the present study, we investigated the relationshipbetween sexual advertisement and immune function in a naturalpopulation of male wolf spiders, Hygrolycosa rubrofasciata (Araneae:Lycosidae). Males of H. rubrofasciata have a costly and condition-dependentacoustic signal, courtship drumming. In the mating season, malesdrum against dry leaves while wandering around the habitat searchingfor receptive females. Males increase their mating success byincreasing their drumming rate and mobility. We used drummingrate and mobility measured without female proximity as estimatesof sexual advertisement. As estimates of male immune function,we used encapsulation rate and lytic activity. Encapsulationrate is a common challenging technique, which measures immuneresponse against multicellular parasites. Lytic activity isa monitoring technique, which measures immune response againstpathogens. Our results show that males with higher drummingrate had higher encapsulation rate. This suggests that femalesmight use drumming rate as a signal for choosing males withgood immunocompetence. Moreover, our results show that maleswith higher mobility had higher lytic activity. As females aremore likely to encounter those males that have higher mobility,this might also select males with better immune function. Ourresults suggest that the immunocompetence handicap might workalso among spiders, although we could not assess the causalityof the relationship between sexual selection and immune functionin this correlational study.