The complete amino acid sequence of the rabbit P2 protein

P2 protein is a small basic protein (Mr = 14,820) found in peripheral nerve myelin and spinal cord myelin. There is now overwhelming evidence that P2 protein is the crucial antigen involved in the induction of experimental allergic neuritis, an autoimmune disease of the peripheral nervous system. The complete amino acid sequence of rabbit P2 protein was derived by sequence analysis of cyanogen bromide peptides and peptides obtained by proteolysis using Staphylococcus aureus V8 enzyme, trypsin, or clostripain. There are 131 amino acids and an excess of the basic amino acids lysine and arginine; histidine is absent. There are 3 highly hydrophobic regions in the P2 molecule. Probability analysis of the sequence predicts a high degree of beta structure, essentially in agreement with CD data.     

The use of acridine orange for testing blood platelet integrity

Two processes are involved in the accumulation of acridine orange in human blood platelets. One follows a diffusion like kinetics and is independent of the ATP level whereas the second one can be completely abolished by ATP depletion. The acridine orange incorporation rate seems to be a suitable parameter for testing platelet integrity. It reflects very sensitively the influence of the preparation method as well as of anticoagulating substances used on the stability of platelet suspensions. The rates of acridine orange incorporation and of aggregation were measured in platelet-rich plasma and in saline suspended platelets after gel filtration, respectively, over a period of 120 min storage. Both rates are influenced to a different degree by anticoagulating agents such as citrate, heparin and EDTA. When contact with anticoagulating agents during platelet preparation is avoided, platelets show a constant acridine orange incorporation and aggregation during storage and the smallest morphological alteration.     

Modulation of histone H5-nucleosome interactions by H5 phosphorylation

In nucleosomal particles of 180 base pairs, part of the histone H5 binding site is preserved. After fluorescein labelling of H5 from chicken erythrocytes comparative equilibrium binding studies have been performed and on these particle as well as on core particles (140 base pairs) and on free DNA (180 base pairs). While nucleosomal particles can accommodate about the same number of H5 molecules as the free DNA derived from it, affinities are decreased by a factor of 3. A further decrease by factors of 3–4 is the consequence of phosphorylating three of the H5 serines: hence phosphorylation should facilitate thermodynamically controlled complexing of red cell chromatin during erythropoiesis. The most dramatic effect of an H5 phosphorylation is a reduction in the binding sites from 56 to 36 nucleotides (free DNA) and an even more pronounced effect upon interacting with nucleosomes which should make the H5-chromatin association sterically favourable. Related studies with protamines from herring are included for comparison.     

Biliary lipid secretion in hypercholesterolemia.

A report on the effects of primary bile acid ingestion alone or in combination with plant sterols on serum cholesterol levels, biliary lipid secretion, and bile acid metabolism. Biliary bile acid and cholesterol secretion were measured in four patients with type IIa hypercholesterolemia before and after randomized treatment periods. During these periods either a bile acid mixture (cholic-chenodeoxycholic 2:1, a proportion similar to that endogenously synthesized in health), at a level of 20 mg/kg, or the same mixture plus sitosterols, 200 mg/kg, was fed. Serum cholesterol and the cholesterol saturation of fasting-state bile was also measured. Pretreatment biliary lipid secretion was within normal limits. Bile acid kinetic measurements were also recorded before treatment and showed that cholic acid synthesis was disproportionately decreased relative to that of chenodeoxycholic acid, a finding previously reported by others. Administration of the bile acid mixture increased biliary bile acid secretion in 3 of 4 patients, but did not influence biliary cholesterol secretion. The combination of sitosterol-bile acid, however, caused a relative decrease in cholesterol secretion in bile, and fasting-state bile became unsaturated in all patients. No change in fecal neutral sterol excretion occurred during the beta-sitosterol-bile acid regimen, suggesting that simultaneous bile acid feeding blocks the compensatory increase in cholesterol synthesis known to be induced by beta-sitosterol feeding in hypercholesterolemic patients. Serum cholesterol levels also fell modestly during the sitosterol-bile acid regimen, the decrease averaging 15%. We conclude that the abnormally low rate of bile acid synthesis in patients with type IIa hyperlipoproteinemia does not influence biliary lipid secretion; that increasing the input of the two primary bile acids into the enterohepatic circulation does not increase biliary cholesterol secretion or lower serum cholesterol levels in such patients; and that the usual increase in cholesterol synthesis induced by beta-sitosterol feeding does not occur if bile acids are administered simultaneously.     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine     

Isolation and purification of bacterial membrane proteins by the use of organic solvents: The lactose permease and the carbodiimide-reactive protein of the adenosinetriphosphatase complex of escherichia coli

Techniques for the solubilization and fractionation of integral membrane proteins have been developed in recent years. A small portion of membrane protein (about 2%, proteolipid fraction) will partition into chloroform or 1-butanol, and, in several cases, these proteins retain functional activity. A virtually complete solubilization can be achieved at neutral pH by use of aprotic solvents, like hexamethylphosphoric triamide or N-methylpyrrolidone. At relatively low concentrations (< 3 M) aprotic solvents inhibited β-D-galactoside transport by whole cells and the derivative membrane vesicles of Escherichia coli, but this inhibition could be largely reversed by a simple washing procedure. At higher concentrations of aprotic solvent (5–6 M), 50–80% of the total protein of lactose transport-positive membrane vesicles was solubilized. When these extracts were added to intact lactose transport-negative membrane vesicles, lactose transport was reconstituted, the required energy being provided by either respiration (e.g., addition of D-lactate) or by a K⁺ diffusion potential established with the aid of valinomycin. The dicyclohexylcarbodiimide (DCCD)-reactive subunit of the E. coli ATPase complex was found to partition into chloroform, and to be amenable to further purification in organic solvent. Ether precipitation and chromatography on DEAE-cellulose and hydroxypropyl-Sephadex G-50 yielded an homogeneous polypeptide of an apparent molecular weight of 9,000. The purified and unlabeled DCCD-reactive protein was incorporated into K⁺-loaded liposomes, and a membrane potential was generated by the addition of valinomycin. There are indications that the DCCD-reactive protein alone made the membrane specifically permeable for protons.     

The influence of chronic ethanol feeding to rats on the integrity of liver mitochondrial membrane as assessed with the Mg2+-stimulated ATPase enzyme.

The mouse submaxillary proteases (A + D), the isolation and properties of which were previously described by us, hydrolyze only arginyl bonds in proteins. Formol titrations and peptide mapping on digests of polyarginine, polylysine, lysozyme, histone, and insulin suggested this specificity. Amino acid compositions of peptides from lysozyme and insulin showed that most but not all arginyl bonds were hydrolyzed but that no lysyl bonds were split. The proteases should be useful in the sequencing of proteins.     

Weichselian chironomid and cladoceran assemblages from maar lakes

Sediments from maar lakes in the periglacial area were analysed to obtain information about the limnetic fauna of Pleniglacial lakes. On the basis of abundance and species composition of the subfossil chironomid and cladoceran assemblages, three major zones were biostratigraphically separated: the Upper Pleniglacial, the Middle Pleniglacial, and the Eem Interglacial.     

Transfer RNA genes from Dictyostelium discoideum are frequently associated with repetitive elements and contain consensus boxes in their 5' and 3'-flanking regions.

A total of 68 different tRNA genes from the cellular slime mold Dictyostelium discoideum have been isolated and characterized. Although these tRNA genes show features common to typical nuclear tRNA genes from other organisms, several unique characteristics are apparent: (1) the 5'-proximal flanking region is very similar for most of the tRNA genes; (2) more than 80% of the tRNA genes contain an "ex-B motif" within their 3'-flanking region, which strongly resembles characteristics of the consensus sequence of a T-stem/T-loop region (B-box) of a tRNA gene; (3) probably more than 50% of the tRNA genes in certain D. discoideum strains are associated with a retrotransposon, termed DRE (Dictyostelium repetitive element), or with a transposon, termed Tdd-3 (Transposon Dictyostelium discoideum). DRE always occurs 50 (+/- 3) nucleotides upstream and Tdd-3 always occurs 100 (+/- 20) nucleotides downstream from the tRNA gene. D. discoideum tRNA genes are organized in multicopy gene families consisting of 5 to 20 individual genes. Members of a particular gene family are identical within the mature tRNA coding region while flanking sequences are idiosyncratic.