Membrane microheterogeneity: Förster resonance energy transfer characterization of lateral membrane domains

Lateral membrane heterogeneity, in the form of lipid rafts and microdomains, is currently implicated in cell processes including signal transduction, endocytosis, and cholesterol trafficking. Various biophysical techniques have been used to detect and characterize lateral membrane domains. Among these, Förster resonance energy transfer (FRET) has the crucial advantage of being sensitive to domain sizes smaller than 50-100 nm, below the resolution of optical microscopy but, apparently, similar to those of rafts in cell membranes. In the last decade, several formalisms for the analysis of FRET in heterogeneous membrane systems have been derived and applied to the study of microdomains. They are critically described and illustrated here.     

Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.     

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function; tests for receptor-stimulated phosphatidylinositol turnover and for modulation of agonist binding by guanylylimidodiphosphate showed functional coupling to be present several days prior to the onset of synapse formation. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed.     

Analysis of the mode of antigen presentation required for triggering of T cell proliferation by using various azobenzenearsonate-tyrosine derivatives

Various azobenzenearsonate-tyrosine (ABA-Tyr) derivatives were synthesized by modifying amino and carboxyl groups at the alpha-carbon of tyrosine, with preservation of most of the ABA-Tyr moiety (ABA plus hydroxyphenyl portion of tyrosine). These derivatives were tested for the ability to stimulate ABA-L-Tyr specific T cell lines derived from B10.BR and B10.S mice. ABA-acetyltyramine, ABA-hydroxyphenylpropionic acid (ABA-PPr), and ABA-propylphenol, which lack either the carboxyl or amino group or both, could not induce T cell proliferation. The lack of stimulation by these derivatives was not due to their cytotoxic effects. A similar pattern of proliferation was obtained on stimulating lymph node T cells from B10.BR and B10.S mice primed with ABA-L-Tyr. Some differences were observed, however, between B10.BR and B10.S mice. ABA-L-Tyr-specific T cells from B10.BR mice could not respond well to ABA-D-Tyr in contrast to B10.S T cells. Furthermore, B10.BR mice primed with ABA-acetyltyramine or ABA-PPr in complete Freund's adjuvant could not induce ABA-L-Tyr-reactive T cells, whereas T cells from B10.S mice primed with these derivatives could proliferate in the presence of ABA-L-Tyr. The differences between B10.BR and B10.S mice were further investigated by using (B10.S X B10.BR)F1 mice. T cells from ABA-L-Tyr-immunized F1 mice responded poorly to ABA-D-Tyr when presented with B10.BR antigen-presenting cells (APC), but responded well when presented with B10.S APC. Similarly, T cells from ABA-PPr-primed F1 mice did not proliferate to ABA-L-Tyr in the presence of B10.BR APC, but could proliferate in the presence of B10.S APC. Our results clearly indicate that the presence of charged groups at the alpha-carbon of tyrosine plays a critical role in the triggering of ABA-L-Tyr-specific T cell proliferation. The significance of these results is discussed.     

Cytotoxicity of cholesterol oxidase to cells of hypercholesterolemic guinea pigs

1. A high cholesterol diet caused guinea pig erythrocytes to become sensitive to lysis by cholesterol oxidase (CO), a protein not hemolytic to normal cells. 2. Lysis was associated with conversion of membrane cholesterol to its oxidation product (delta-4-cholesten-3-one). 3. Intravenous injection of CO to hypercholesterolemic guinea pigs produced a reduction in serum cholesterol, but was not lethal as it was in rabbits. 4. Homogenized spleen, liver and kidney from the hyperlipidemic animals were sensitive to in vitro cholesterol oxidation while tissues from non-lipemic animals were resistant to modification.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Potentiation by naltrexone of d-amphetamine-induced behavioral suppression and its reversal by clonidine

Rats were trained to perform a fixed ratio-15 operant for food reinforcement during a 30 minute daily session. Naltrexone, in doses up to 45 mg/kg administered 15 min before the behavioral session, failed to disrupt responding. However, 0.3 and 1.0 mg naltrexone/kg produced a dose related potentiation of the operant behavioral suppression induced by 1.0 mg d-amphetamine/kg injected immediately before the session. The naltrexone/d-amphetamine combination also produced excessive salivation and postural abnormalities not seen when either drug was administered alone. [A subsequent study indicated that the salivation induced by naltrexone in combination with d-amphetamine may require previous exposure to naltrexone and/or d-amphetamine.] Blockade of dopamine receptors with pimozide did not modify the interaction. Functional noradrenergic blockade with a low dose of clonidine significantly reversed the potentiated suppression, of operant behavior, as well as the excessive salivation and abnormal posture. These data suggest that there is an important noradrenergic component to the interaction of naltrexone with d-amphetamine. The impressive interaction of behaviorally inactive doses of naltrexone with a moderate dose of d-amphetamine reported here for rats may have clinical implications for detoxified opiate addicts maintained on naltrexone in antagonist therapy programs.     

Frequency of Escherichia coli O157:H7 isolation from stool specimens

During a 6-month period, 7252 faeces specimens were examined for Escherichia coli serotype O157:H7. Forty-nine specimens (0.7%) from 19 patients yielded this organism. Escherichia coli O157:H7 was the third most frequently isolated bacterial pathogen, following Campylobacter jejuni and (or) Salmonella sp. Although regional variation between laboratories determined whether Campylobacter jejuni or Salmonella was the primary bacterial pathogen isolated, E. coli O157:H7 was consistently isolated more frequently than either Shigella or Yersinia enterocolitica.