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.     

Hormonal regulation of the alpha-ketoglutarate dehydrogenase complex in the isolated perfused rat liver

The metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in perfused livers was monitored by measuring the rate of 14CO2 production from [1-14C]alpha-ketoglutarate. The rates of 14CO2 production and glucose production from [1-14C]alpha-ketoglutarate were increased with increasing perfusate alpha-ketoglutarate concentrations. Vasopressin, angiotensin II, and the alpha 1-adrenergic agonist phenylephrine stimulated transiently by 2.5-fold the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction in the presence and absence of Ca2+ in the perfusion medium. High concentrations of glucagon (1 x 10(-8) M) and 8-p-chlorophenylthio-cAMP (100 microM) (data not shown) also stimulated transiently the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction. However, lower glucagon concentrations (1 x 10(-9) M) stimulated the rate of 14CO2 production from [1-14C]alpha-ketoglutarate only under conditions optimized to fix the cellular oxidation-reduction state at an intermediate level, when glucagon (1 x 10(-9) M)-mediated elevation of cAMP content was greater than that observed under highly oxidizing and reducing conditions. These data indicate that agonists which increase cytosolic free Ca2+ levels stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase complex. Furthermore, the data presented here demonstrate for the first time that physiological glucagon concentrations stimulate the metabolic flux through the alpha-ketoglutarate dehydrogenase reaction only under conditions known to be optimal for glucagon-mediated Ca2+ mobilization in the isolated perfused rat liver.     

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.     

Boring Forminifera in French Polynesian coral reefs

The discovery of epipsammitic Foraminifera in sediments of Moorea and Scilly (French Polynesia) and the study of close relationships between embedded specimens and host-grains indicate that some species participate in the weakening and subsequent breakdown of skeletal grains and consequently contribute to the production of silt-sized particles. The study of 36 stations around the islands shows the factors that control the distribution and abundance of this incrusted microfauna. Specimens are more abundant on sand particles larger than 1,000 microns and in the high energy areas; this abundance decreases with depth. The physiological mechanism of penetrations is presumably chemical, but each species apparently has its own process: complete dissolution with removal of ions for the cytoplasm of calcareous species; partial dissolution with transport of silt-size aggregates on the test of agglutinated species. The purpose of such penetration may be to protect themselves against water turbulence and to provide material for test construction.     

Evolution of the immunodominant domain of the circumsporozoite protein gene from Plasmodium vivax. Implications for vaccines

Recent work directed toward the development of a malarial vaccine has focused on the identification and production of the immunodominant repeating peptide of the circumsporozoite protein of the human malaria parasites as an antigen. An important factor which relates to the usefulness of this antigen in a vaccine is the rate at which the molecule changes in sequence. We have determined the sequence and arrangement of the repeating epitope of the circumsporozoite protein gene from a Plasmodium vivax isolate from La Paz, El Salvador (Sal-I). This is compared with a portion of the previously published sequence of the circumsporozoite protein gene from a P. vivax isolate from Belém, Brazil. The genes appear to be very similar in the repeat region. There are 20 similar repeating units in the El Salvador strain and only 19 units are conserved in the Brazilian strain. Following this there are degenerate repeats in both strains. Even the pattern of silent mutations in the repeat area are similar; however, they are not necessarily in the identical location and appear to have shifted. The data suggest that the repeat region of these genes may be evolving by an accelerated mechanism(s). Such a phenomenon could severely decrease the long-term efficacy of a repeat-based anti-sporozoite vaccine.     

Origin of abnormality in a human simelian foetus as elucidated by our knowledge of vertebrate development

In this paper we attempt to explain the abnormality of a simelian foetus with reference to our present knowledge of vertebrate development. The various developmental defects seem to have a single common origin: the speeding-up of the progression of cell differentiation in the notochord anlage--which is the organization centre of the embryo--during the regression of the Hensen's node. Cell activity involved in the morphogenetic movements in the chordamesoderm probably stopped before it should have. The elongation of the notochord anlage was not completed, resulting in the defective development of the posterior part of the foetus. A number of pairs of posterior trunk somites were not induced. Consequently (1) the pelvic limb buds, whose posterior parts were missing, fused, bringing in further developmental deviations in the limb skeleton and abdominal muscles; (2) there are no vertebrae between the first sacral vertebra and the misshaped coccyx formed by the tail bud. The derivatives of the posterior endoderm (hindgut, bladder and ureters) were not induced either. The cauda equina is deficient. The absence of functional kidneys and the presence of embryonic urinary tubules in the pelvic cysts which are wrapped up by gut epithelium suggest the induction of the metanephric mesenchyme by ectopic endoderm. The speeding-up of differentiation in the notochord anlage also probably resulted in the excessive extension of its anterior region which is the organizer of brain structures. This explains the overdevelopment of the nose and of the neurocranium, and the low position of the ear. A gene mutation as well as a mechanical stress are the possible causes of the abnormal behaviour of the notochord anlage.