Age related changes in lipid peroxidation in rat brain and liver

Lipid peroxidation in rat liver, unlike in brain shows wide variations with age. In liver, ascorbic acid content also undergoes wide variations and there is negative correlation between ascorbic acid content and lipid peroxidation. Heat-labile antioxidant factors are present in the cytosol fraction. There is inverse relationship between antioxidant activity and lipid peroxidation in liver.     

Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues

Epithelial cells contain a cytoskeletal system of intermediate-sized (7 to 11 nm) filaments formed by proteins related to epidermal keratins (cytokeratins). Cytoskeletal proteins from different epithelial tissues (e.g. epidermis and basaliomas, cornea, tongue, esophagus, liver, intestine, uterus) of various species (man, cow, rat, mouse) as well as from diverse cultured epithelial cells have been analyzed by one and two-dimensional gel electrophoresis. Major cytokeratin polypeptides are identified by immunological cross-reaction and phosphorylated cytokeratins by [³²P]phosphate labeling in vivo.It is shown that different epithelia exhibit different patterns of cytokeratin polypeptides varying in molecular weights (range: 40,000 to 68,000) and electrical charges (isoelectric pH range: 5 to 8.5). Basic cytokeratins, which usually represent the largest cytokeratins in those cells in which they occur, have been found in all stratified squamous epithelia examined, and in a murine keratinocyte line (HEL) but not in hepatocytes and intestinal cells, and in most other cell cultures including HeLa cells. Cell type-specificity of cytokeratin patterns is much more pronounced than species diversity. Anatomically related epithelia can express similar patterns of cytokeratin polypeptides. Carcinomas and cultured epithelial cells often continue to synthesize cytokeratins characteristic of their tissue of origin but may also produce, in addition or alternatively, other cytokeratins. It is concluded: (1) unlike other types of intermediate-sized filaments, cytokeratin filaments are highly heterogeneous in composition and can contain basic polypeptides: (2) structurally indistinguishable filaments of the same class, i.e. cytokeratin filaments, are formed, in different epithelial cells of the same species, by different proteins of the cytokeratin family; (3) vertebrate genomes contain relatively large numbers of different cytokeratin genes which are expressed in programs characteristic of specific routes of epithelial differentiation; (4) individual cytokeratins provide tissue- or cell type-specific markers that are useful in the definition and identification of the relatedness or the origin of epithelial and carcinoma cells.     

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH₄NO₃ on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH₄NO₃ decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [³⁵S]sulfate into the components I and II of nitrogenase was similarly not affected.The addition of NH₄NO₃ decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Characterization of microsomal electron transport components from control, phenobarbital- and 3-methylcholanthrene-treated mice. III. Improved resolution and quantitation of major components in ammonium sulfate fractions from total liver microsomes

Quantitation of microsomal components in ammonium sulfate fractions using a high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, and a comparison of these results with those from similar experiments on total liver microsomes has enabled us to identify and better characterize the interactions between microsomal electron transport components.

It was found that: (1) phenobarbital decreased the amount of one protein component of approximately 50 000 molecular weight while increasing a component of very similar molecular weight; (2) only two proteins appeared to be associated with CO binding; (3) another protein of approximately 68 000 molecular weight, one of the glycoproteins found in liver microsomes, appears to be induced by phenobarbital pretreatment; (4) the induction of NADPH-cytochrome c reductase activity after phenobarbital pretreatment is not dependent on an increase in the known NADPH-dependent flavoprotein, but rather on the increase in some component found predominately in our most soluble sub-microsomal fraction.

A very good separation of the above components was achieved by ammonium sulfate fractionation, e.g. simply on the basis of their solubility. This and the fact that the more-or-less soluble proteins were induced by phenobarbital or 3-methylcholanthrene respectively indicate that the solubility of membrane proteins plays a major role in the structure and function of microsomal membranes.     

Changes in the reactivity of proteins of rat liver ribosomes against [C]iodoacetamide depending on their organization in ribosomal subparticles, 80S ribosomes and on the attachment of poly-(U)

(1) The isolated mixtures of ribosomal proteins can be substituted by [¹⁴C]-iodoacetamide up to an average of about 2 equivalents per 20 000 dalton. The extent of substitution of single proteins measured after two-dimensional polyacrylamide gel electrophoresis shows that all proteins are reactive.

(2) Also in the subunits, all proteins are accessible to substitution. Compared with isolated proteins, however, the reactivity is decreased and the amount of labelling for most proteins ranges as low as 5 to 20%.

(3) Reassociation of ribosomal subunits decreases the reactivity of 12 proteins of the small subunit and that of 20 proteins of the large subunit.

(4) The presence of messenger inhibits the substitution of 10 proteins of the small subunit and of 6 proteins of the large one.

(5) Seven proteins of the small subunit and 3 proteins of the large one are influenced both by the other subunit and by messenger-RNA.     

Effect of rate-limiting elongation on bacteriophage MS2 RNA-directed protein synthesis in extracts of Escherichia coli

The consequences of limiting the rate of elongation of protein synthesis in vitro have been examined. The concentration of Trp-tRNA^Trp was manipulated by varying the amount of exogenously added tryptophan in extracts from an Escherichia coli mutant in which the tryptophanyl-tRNA-synthetase has a higher K_M for tryptophan. The evidence presented supports the hypothesis that variation of the rate of elongation can be a means of regulating gene expression, both directly, by slowing or accelerating the rate of protein synthesis and indirectly, by leading to varying three-dimensional structures of the messenger RNA when progress of the ribosomes is perturbed. The data can be described by assuming that if a specific transfer RNA is limiting, to a first approximation the overall rate of protein synthesis is determined by the relative rate of reading past an individual codon requiring that tRNA raised to the power of how many times that codon appears in the message. This could be explained by a model in which, with a significant probability, the ribosome stops protein synthesis prematurely at these codons, falls off the messenger RNA and is available for further rounds of protein synthesis. In agreement with other work, evidence is also presented that suggests that under the most drastic available limitation of the elongation rate, that is, starvation for a given amino acid, reading through the corresponding “hungry codon” occurs in vitro at a surprisingly high rate, possibly due to mistranslation.     

The effect of periodate oxidation and α-mannosidase treatment of Dolichos biflorus lectin

The effects of periodate and α-mannosidase treatment of the Dolichos biflorus lectin were determined. Destruction by periodate of 16% of the mannose residues of the lactin had no effect on its ability to agglutinate type A erythrocytes, precipitate blood group A + H substance or to be precipitated by concanavalin A. Removal of up to 40% of the mannose by either periodate or α-mannosidase rendered the lecton nonprecipitable by concanavalin A. The lectrin treated by α-mannosidase retained its ability to agglutinate erythrocytes and precipitate blood group A + H substance, but the lectin treated with periodate lost most of its activity.The results suggest that the complete integrity of the carbohydrate unit of the lectin is not necessary for its activity and that the periodate may be affecting the protein portion of the molecule as well as its carbohydrate residues. No conversion of form A to form B of the lectin was observed with either periodate oxidation or α-mannosidase treatment.     

Regulation of C4 photosynthesis: regulation of pyruvate, Pi dikinase by ADP-dependent phosphorylation and dephosphorylation

Pyruvate, Pi dikinase in extracts of chloroplasts from mesophyll cells of Zea mays is inactivated by incubation with ADP plus ATP. This inactivation was associated with phosphorylation of a threonine residue on a 100 kDa polypeptide, the major polypeptide of the mesophyll chloroplast stroma, which was identified as the subunit of pyruvate, Pi dikinase. The phosphate originated from the beta-position of ADP as indicated by the labelling of the enzyme during inactivation in the presence of [beta-32P]ADP. During inactivation of the enzyme up to 1 mole of phosphate was incorporated per mole of pyruvate, Pi dikinase subunit inactivated. 32P label was lost from the protein during the Pi-dependent reactivation of pyruvate, Pi dikinase.     

Regulation of glucose-6-p dehydrogenase synthesis in rat epididymal fat pads.

The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis.     

rII cistrons of bacteriophage T4. DNA sequence around the intercistronic divide and positions of genetic landmarks

An 873 base-pair DNA sequence from the rII region of bacteriophage T4 is presented. The sequence encodes 139 carboxyl-terminal amino acids of rIIA and the amino-terminal 146 amino acids of rIIB. Eleven base-pairs separate the rIIA stop codon (UAA) and the rIIB AUG.An extensive genetic map is superimposed on the DNA sequence, showing the deduced locations of many of the mutations (base-pair substitutions, frameshifts, deletions) found in previous rII genetic studies.     

Microtubule reassembly in vitro of Strongylocentrotus purpuratus sperm tail outer doublet tubulin

Strongylocentrotus purpuratus outer doublet microtubules were prepared by extraction of sperm tail axonemes with 0.6 m-KCl. Sonication of the outer doublet microtubules in 5 mm-2-(N-morpholino)ethanesulphonic acid, 1 mm-ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, 1 inm-MgSO₄ (pH 6.7) solubilized up to 35% of the outer doublet protein, depending on the power input, in a manner which was non-selective for either subfiber. Tubulin comprised 75 to 85% of the total solubilized protein in a 200,000 g supernatant obtained from the sonicated suspension. Colchicine-binding assays demonstrated that the tubulin was largely in a native form (K_A = 10⁶, liters mole^?; 0.74 mole of colchicine bound per mole of tubulin at infinite concentration of colchicine).Microtubule self-assembly from the 200,000 g supernatants in the absence of added seeds or glycerol was quantitated by light-scattering at 350 nm. The critical protein concentration for assembly was 0.55 mg ml^?1 at 37 °C and the reaction occurred optimally in the presence of 2 mm-GTP and 150 mm-KCl. The solubilized outer doublet tubulin formed singlet microtubules upon reassembly under our in vitro conditions. The authenticity of the microtubules was verified by both negative stain and thin-section electron microscopy. Polymerization was prevented by colchicine and podophyllotoxin, and depolymerization occurred rapidly on cooling the microtubules to 0 °C.The susceptibility of the reassembled microtubules to low temperature suggested that they could be “recycled” by the warm assembly-cold disassembly procedure developed for vertebrate brain (Borisy et al., 1974). Twice recycled outer doublet tubulin was devoid of high molecular weight microtubule-associated proteins, as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. However, trace amounts (less than 5%) of intermediate molecular weight material was visible on heavily overloaded gels. The function of this material is uncertain, but it is not chemically equivalent to the tau factor of vertebrate brain (Weingarten et al., 1975), since it cannot be separated from the tubulin by phosphocellulose adsorption. In addition, phosphocellulose-treated tubulin reassembled to the same extent as untreated tubulin, suggesting that the reassembly of outer doublet tubulin does not require the protein equivalents of brain microtubule-associated proteins or tau factor. If accessory proteins are required for the reassembly of outer doublet tubulin, they are not removed by phosphocellulose under the conditions employed, and they must comprise less than 5% of the total protein.     

Preparation of membrane vesicles from isolated myelin. Studies on functional and structural properties

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in ²²Na⁺ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myelin basic protein from 0–150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.