Topology of proteolipid protein in the myelin membrane of central nervous system. A study using antipeptide antibodies

Peptides according to amino-acid sequences of the N- and C-terminus of lipophilin (proteolipid protein, PLP) (Gly1-Phe15 = 1; Thr261-Phe276 = 6) and of the other four hydrophilic domains (Glu37-Leu60 = 2; Arg97-Leu112 = 3; Gly119-Gly127 = 3A; Trp144-Tyr156 = 3B; Lys191-Ala203 = 4; Asn222-Phe232 = 5) have been synthesized by the solid-phase Fmoc method, linked covalently to keyhole limpet hemocyanin (KLH) and used as antigens. Monospecific antibodies against these antigens were isolated by affinity chromatography. Each antibody recognized its epitope in isolated partially delipidated PLP with the ELISA technique, western blot, thin sections of paraffin embedded rat brains and in the plasma membrane of appropriately fixed/permeabilized rat oligodendrocytes in culture. After fixation with formaldehyde antipeptide 3A antibody stained intact non-permeabilized cells. Therefore the epitope 3A must be located on the extracellular surface of the membrane. This is in full support of our previous biochemical results on the orientation of lipophilin in the myelin membrane.     

The primary structure of bovine brain myelin lipophilin (proteolipid apoprotein)

The amino-acid sequence of bovine myelin lipophilin (proteolipid apoprotein, Folch-protein) has been completed. Lipophilin is a 276 amino acid residues containing, extremely hydrophobic membrane protein with molecular mass 30,000 Da. The sequence determination was based on automated Edman degradation of four tryptophan and four cyanogen bromide fragments and of proteolytic peptides of complete lipophilin as well as the fragments obtained by chemical cleavage. Four additional sequences were determined which led to the completion of the primary structure. Lipophilin is esterified at threonine-198 by long chain fatty acids (palmitic, stearic and oleic acid). The attachment site has been established at the same threonine residue in three different peptides isolated from thermolysinolytic, papainolytic and chymotrypsinolytic hydrolysates. This threonine residue is part of a hydrophilic segment of lipophilin. The covalent fatty acyl bond is being discussed together with important structural and functional properties of this membrane protein which can be derived from sequence information. New separation and purification methods of hydrophobic and hydrophilic polypeptides for this sequence determination (fractional solubilization, silica gel exclusion, high-performance liquid chromatography) had to be elaborated as indispensable tools. They are generally applicable to the structural analysis of hydrophobic membrane proteins. Four long (26, 29, 40 and 36 residues) and one medium long (12 residues) hydrophobic segments are separated by four predominantly positively and one negatively charged hydrophilic segments. On the basis of structural data a model for the membrane integration of lipophilin is proposed.     

Studies on the asymmetric arrangement of membrane-lipid-enveloped virions as a model system.

Lipids of BHK 21 cells (baby hamster kidney) grown in tissue culture were labelled with radioactive fatty acids. The enveloped vesicular stomatitis virus was propagated in this host cell type. The virions were purified by density gradient centrifugation. Neuraminidase treatment of the intact virions led to a complete transformation of hematoside [N-acetylneuraminosyl(alpha2-3)lactosyl(beta1-1)ceramide] into lactosylceramide, with identical labelling of the ceramide portion in hematoside of the untreated virions and the lactosylceramide of the neuraminidase-treated particles. The morphology of the virions appeared unchanged in electron micrographs, but the neuraminic-acid-free virions had a strong tendency to aggregate. The results of these studies are evidence that gangliosides are integrated exclusively into the outer lamella of the lipid bilayer in the viral envelope. It is also evident that the viral envelope is a suitable model for studies on membrane asymmetry.     

13-C nuclear magnetic resonance studies on the lipid organization in enveloped virions (vesicular stomatitis virus).

13-C nuclear magnetic resonance (NMR) studies are described regarding the lipid organization in the envelope of the vesicular stomatitis virion. The fatty acid chains (oleic acid) and the choline moiety of the 3-sn-phosphatidylcholine and spingomyelin have been labeled specifically with 13-C by growing the virions in prelabeled host cells (BHK 21 cells). The results suggest that 130C NMR spectroscopy is a very feasible method for the study of natural membranes provided the isotope is highly enriched in specific positions and incorporated biochemically. Spin-lattice relaxation (T1) measurements of particular C atoms have been carried out with whole virions, with virions deprived of their surface projections by trypsinization but unaltered in their shape and size, and with liposomes prepared from the total lipid mixture of the envelope in order to get insight into the molecular structure of this model membrane. The mobility of the central part of 11-13-C-labeled oleic acid incorporated into the ester and amide lipids and the choline group of 3-sn-phosphatidylcholine and sphingomyelin is very restricted as indicated by their short T1 times. It is concluded from the data presented here that the high cholesterol content (cholesterol/P: 0.7) of the envelope lipid phase is responsible for the rather rigidly packed envelope structure. The mode and extent of the interactions between lipids and glycoprotein surface projections are subjects for further study.     

K+ Efflux and Retention in Response to NaCl Stress Do Not Predict Salt Tolerance in Contrasting Genotypes of Rice (Oryza sativa L.)

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K⁺) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with ⁴²K, coupled with growth and tissue K⁺ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K⁺ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K⁺ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K⁺ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na⁺ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K⁺ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K⁺ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na⁺ accumulation is the key factor in performance decline on NaCl stress.     

A journey through the Amazon Middle Earth reveals Aspidoras azaghal (Siluriformes: Callichthyidae), a new species of armoured catfish from the rio Xingu basin,Brazil

Aspidoras azaghal n. sp. was discovered during a multitaxonomic scientific expedition to the remote Amazon Terra do Meio region in tributaries to the rio Xingu basin, Pará, Brazil. The new species can be promptly distinguished from its congeners by the following combination of features: (a) absence of the first dorsal-fin element; (b) parieto-supraoccipital fontanel located medially on bone; (c) absence of a longitudinal dark-brown or black stripe along flank midline; (d) ventral surface of trunk covered by clearly smaller, irregular and/or roundish platelets; (e) inner laminar expansion of infraorbital 1 well developed; (f) relatively wide frontal bone, with width equal to half of entire length; (g) absence of a thick, longitudinal conspicuous dark-brown stripe along dorsal portion of flank; and (h) poorly developed serrations on posterior margin of the pectoral-fin spine. Besides morphological evidence, the molecular analyses indicated significant differences between the new species and its congeners, with A. albater and A. raimundi as its closest species, showing 6.53% of genetic differentiation in both cases. The intraspecific molecular data revealed gene flow (peer fixation index, FST = 0.05249, P > 0.05, for the cytochrome oxidase I (COI) marker and FST = -0.01466, P > 0.05, for the control region) between specimens upstream and downstream from a 30-m height waterfall at the type-locality, which therefore represent a single population. Furthermore, it was possible to observe a unidirectional gene flow pattern, with genetic diversity increasing in the downstream direction.     

The local electrostatic environment determines cysteine reactivity of tubulin

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide, N-[(14)C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity.