DISTRIBUTION OF POLYSOMES IN MOUSE BRAIN TISSUE

Abstract— Polysomes were isolated from several different fractions of mouse brain tissue. After homogenization, the extract was centrifuged to yield a post-mitochondrial supernatant fraction and a pellet fraction. Sucrose gradient analysis of the material in the post-mitochondrial supernatant fraction indicated that 80 per cent of the ribosomes were present in polysomes and that little, if any, of the pellet fraction was present. Sucrose gradient analysis of the solution obtained after washing the pellet showed that very little polysomal material was present. The remaining pellet fraction was resuspended in a detergent mixture of deoxycholate-Tween 40. Sucrose gradient analysis of the resulting detergent-soluble solution indicated that large amounts of ribosomal material, in which 60–70 per cent of the ribosomes were associated in polysomes, were present.
In brain tissue from young animals, 20 per cent of the polysomes were found in the post-mitochondrial supernatant fraction whereas 80 per cent of the polysomes were released from the pellet fraction by detergent treatment. In contrast, in brain tissue from adult animals, 40 per cent of the polysomes were found in the post-mitochondrial supernatant fraction, whereas 60 per cent of the polysomes were released from the pellet fraction by detergent treatment.     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.     

Fractionation and Characterization of the Plasma and Mesosome Membrane of Listeria monocytogenes

Protoplasts of Listeria monocytogenes strain 42 were fractionated after control lysis on a Ficoll (a polysucrose) density gradient. Visually, five zones could be recognized in the gradient. The first one was composed of amorphous cytoplasmic solutes (fraction 1a) and a mixture of particles (fraction 1b). These were: (i) light particles that were lipase-sensitive and composed of six subunits and (ii) heavy particles, sensitive to ribonuclease and devoid of fine structure. The second zone consisted of tubules and vesicles still harboring cytoplasmic components (fraction 2), whereas the third zone contained only empty vesicles and protoplast ghosts (fraction 3). The material congregating into the fourth zone was morphologically identical to that of the third (fraction 3a). The fifth and heaviest zone contained a mixture of (i) particles without any substructure and (ii) partly lysed protoplasts (fraction 4). Fractions 1b and 4 were the richest in nucleic acids (ribonucleic acid, 11.4 and 9.4%, respectively; deoxyribonucleic acid, 5.1 and 4.8%, respectively), whereas fraction 1b had the highest protein contents (74.6%). Phospholipids were mainly found in fractions 2 and 3. Except for fraction 1, all materials contained significant amounts of protein-bound phosphorus. The main concentrations of four enzymes were: glucose-6-phosphate dehydrogenase (fraction 1a); adenosine triphosphatase and reduced nicotinamide adenine diphosphate oxidase (fraction 3); nitro blue tetrazolium chloride reductase (fraction 2). Fractionation of strain 42 after addition of (32)P during the mid-log phase of growth revealed that the radio-activity was mainly detected in fraction 1b, when growth in the presence of the marker was allowed for 10 min, and in fraction 2, when growth was allowed for 90 min. The vesicles of fraction 2, often tubular, are probably of mesosomal origin, whereas those of fraction 3, which are always spherical, represent, most likely, the bulk of the cell plasma membrane. Our data showed slight chemical differences between these two fractions, but the differences in enzymatic activities and lipid-phosphorus incorporation during long pulse experiments were most dramatic.     

Protein synthesis by brain-cortex mitochondria. Characterization of a 55S mitochondrial ribosome as the functional unit in protein synthesis by cortex mitochondria and its distinction from a contaminant cytoplasmic protein-synthesizing system

Homogenates of rat brain cortex were fractionated by conventional methods of velocity sedimentation and separated into a microsomal and a washed mitochondrial fraction. By electron microscopy the mitochondrial fraction was shown to be rich in synaptosomes. The mitochondria-synaptosome fraction synthesized protein in vitro by a route that was partially inhibited by cycloheximide and partly by chloramphenicol. The relative effectiveness of the two inhibitors varied greatly with the medium used. In the mitochondria-synaptosome fraction active 80S cytoplasmic ribosomes and active 55S mitochondrial ribosomes were detected; these were also seen in the electron microscope. Mild osmotic shock of the mitochondria-synaptosome fraction followed by velocity sedimentation in sucrose-EDTA allowed isolation of a mitochondrial fraction free of synaptosomes. Protein synthesis in this fraction was entirely inhibited by chloramphenicol, but was completely resistant to cycloheximide both in a medium promoting oxidative phosphorylation and in ATP-generating medium. Ouabain had no inhibitory effect on protein synthesis in a purified mitochondrial preparation. It is concluded that brain-cortex mitochondria synthesize protein entirely on 55S mitochondrial ribosomes.     

Differential inhibitory effects of carbon tetrachloride on the hepatic plasma membrane, mitochondrial and endoplasmic reticular calcium transport systems: implications to hepatotoxicity

Mitochondrial, endoplasmic reticular and plasma membrane fractions were isolated by a new method from control male Fischer 344 rats and rats given CCl4 by gavage. After 1 h of CCl4 treatment, rats were in glucose and pancreatic hormone balance but plasma levels of T3 and T4 were decreased 29 and 22%, respectively. After 24 hours of CCl4 treatment, rats were: hypoglycaemic and insulin and glucagon levels were increased 33- and 35-fold, respectively; total T4 levels were decreased 62%; while total T3 levels were normalized. In liver fractions from CCl4-treated rats, 1 h after CCl4 administration: (i) calcium binding was decreased 65% in the mitochondrial fraction, 66% in the endoplasmic reticular fraction and 46% in the plasma membrane fraction; (ii) calcium uptake was decreased 59% in the mitochondrial fraction, 46% in the endoplasmic reticular fraction and 37% in the plasma membrane fraction. After 24 h of CCl4 administration: (i) calcium binding was decreased 57% in the mitochondrial fraction, 50% in the endoplasmic reticular fraction and 71% in the plasma membrane fraction; (ii). calcium uptake was decreased 55% in the mitochondrial fraction, 17% in the endoplasmic reticular fraction and 53% in the plasma membrane fraction. In vitro studies indicated the plasma membrane calcium transport system to be rapidly (within a minute) and strongly (>90%) inhibited by CCl4. We conclude that CCl4 produces a differential inhibitory effect on the hepatocyte calcium pumps that are implicated with hepatocellular damage.     

Accuracy of cancellous bone volume fraction measured by micro-CT scanning

Volume fraction, the single most important parameter in describing trabecular microstructure, can easily be calculated from three-dimensional reconstructions of micro-CT images. This study sought to quantify the accuracy of this measurement. One hundred and sixty human cancellous bone specimens which covered a large range of volume fraction (9.8-39.8%) were produced. The specimens were micro-CT scanned, and the volume fraction based on Archimedes' principle was determined as a reference. After scanning, all micro-CT data were segmented using individual thresholds determined by the scanner supplied algorithm (method I). A significant deviation of volume fraction from method I was found: both the y-intercept and the slope of the regression line were significantly different from those of the Archimedes-based volume fraction (p < 0.001). New individual thresholds were determined based on a calibration of volume fraction to the Archimedes-based volume fractions (method II). The mean thresholds of the two methods were applied to segment 20 randomly selected specimens. The results showed that volume fraction using the mean threshold of method I was underestimated by 4% (p = 0.001), whereas the mean threshold of method II yielded accurate values. The precision of the measurement was excellent. Our data show that care must be taken when applying thresholds in generating 3-D data, and that a fixed threshold may be used to obtain reliable volume fraction data. This fixed threshold may be determined from the Archimedes-based volume fraction of a subgroup of specimens. The threshold may vary between different materials, and so it should be determined whenever a study series is performed.     

Aspects of the protein and the lipid composition of myelinoid marchi-positive bodies from mammalian spinal cord

The fraction floating on 0.32 M sucrose was isolated from normal mammalian spinal cord and analyzed with regard to protein and lipid composition. Comparisons were made with the myelin fraction isolated from the same spinal cord. A close relationship between the two fractions was indicated by a similar protein banding on SDS-polyacrylamide gel electrophoresis. The relative amounts of various proteins however were different and some high molecular weight proteins appeared unique to the floating fraction. The phospho- and galactolipid patterns, as revealed by thin-layer chromatography, were similar in the floating and the myelin fractions. The proportion of hydrophobic lipids, such as sterols and isoprenyl derivatives, was higher in the floating fraction. Bands co-migrating with cholesterol esters were detected only in the floating fraction from guinea pigs. Marchi-positive material of possible paranodal origin is enriched in the floating fraction. The present findings of a biochemical composition of the floating fraction closely resembling that of myelin is in line with the view that myelin turnover includes a step of degradation localized to the paranodal regions.     

Effect of pheromone components when applied to different models on male sexual behaviour in the housefly,Musca domestica

Male mating behaviour was studied by treating a pseudofly, newly emerged female or male fly, with all possible combinations of (Z)-9-tricosene, the non-hydrocarbon fraction and the methylalkane fraction. All dosages were at physiological levels. Striking behaviour, both mating and homosexual, were increased by tricosene. When the non-hydrocarbon fraction was added to the tricosene, homosexual strikes decreased while mating strikes increased. This indicates that the non-hydrocarbon fraction contains sex recognition factors. Increased copulatory attempts were observed with the non-hydrocarbon fraction and the methylalkane fraction but not with the tricosene. The amount of time spent with the model, an arrestant effect, was determined by the methylalkane fraction.     

Cellular daunomycin fluorescence in multidrug resistant 2780AD cells and its relation to cellular drug localisation

Multidrug resistant (MDR) 2780AD human ovarian carcinoma cells were loaded with the fluorescent anticancer agent daunomycin (DN). Fluorescence anisotropy was lower than for corresponding A2780 wild-type cells, indicating that DN was less rigidly bound than in the wild-type cells. Average fluorescence quenching of DN was lower for 2780AD cells. Data were fitted into a model with a highly quenched fraction (fraction A), corresponding to DN intercalated in DNA, and an unquenched fraction (fraction B). The ratio A/B was one order of magnitude lower for the MDR cells than for the wild-type cells. Two other MDR cell lines were investigated and low A/B ratios were found in both cases. Thus, evidence has been provided that in MDR cells the DNA-bound fraction is relatively low and that more free DN is present, for example in acidic vesicles.     

SYNTHESIS OF ACETYLCHOLINE IN DIFFERENT COMPARTMENTS OF BRAIN NERVE TERMINALS IN VIVO AS STUDIED BY THE INCORPORATION OF CHOLINE FROM PLASMA AND THE EFFECT OF PENTOBARBITAL ON THIS PROCESS

The time course of the incorporation of choline from plasma into a high and a low molecular weight fraction from mouse brain synaptosomes was studied. The fractions were obtained from lysed synaptosomes by gel filtration on Sephadex G-25. An extremely rapid incorporation of radioactivity into acetylcholine was found in both fractions and in the time interval 0.25-9 min after the intravenous administration of labelled choline, higher specific radioactivities of acetylcholine were found in the high molecular weight fraction than in the low molecular weight fraction. However, the specific radioactivity of choline in the high molecular weight fraction was much lower than that of acetylcholine. It was found that barbiturate anaesthesia caused a marked decrease in the labelling of acetylcholine in the high molecular weight fraction while the incorporation into the low molecular weight fraction was affected to a much smaller extent. Acetylcholine of the high molecular weight fraction showed properties similar to those of vesicle-bound acetylcholine. The recoveries of labelled and endogenous acetylcholine and choline from the brain homogenates were calculated in different steps of the fractionation procedure. In the fraction containing lysed synaptosomes the recovery of radioactive acetylcholine was lower than that of endogenous acetylcholine. This may indicate the presence of two types of bound acetylcholine in the synaptosomes. Different models for the intraneuronal synthesis of acetylcholine are discussed and it is proposed that a site of acetylcholine synthesis in vivo may be closely associated with some constituent of the high molecular weight fraction and directly coupled with the storage of the transmitter.     

Involvement of a Cell Envelope Component of Escherichia coli in the Early Stages of Infection with Bacteriophage ϕX174

Envelope fraction I prepared from a ?X174 sensitive host, KD4301, showed a strong eclipsing activity, while the lipopolysaccharide (LPS) fraction showed a weak activity. The eclipsing activity in envelope fraction I was sensitive to heat treatment, while that in the LPS fraction was insensitive. When the complete phage particles (114S) were treated with envelope fraction I, the eclipsed particles (70S) and a rapidly sedimenting component were obtained, but when they were treated with LPS, only 70S eclipsed particles were obtained. Electron microscopic observation showed that there were two types of eclipsed particles formed on treatment with fraction I; in one of them phage DNA was extruded from the phage particles as a thick bundle, and in the other more than 95% of the phage DNA was extruded from the phage particles. The rapidly sedimenting component was the membrane-eclipsed particle complex. LPS gave only one type of eclipsed particles in which DNA was extruded as a thick bundle. These results indicate that a heat labile component in the cell envelopes other than LPS is involved in the extrusion of ?X174 DNA.     

Biological activities of lipopolysaccharide fractionated by preparative acrylamide gel electrophoresis

Lipopolysaccharide from a smooth strain of Salmonella minnesota was fractionated into two major fractions and one intermediate fraction by using sodium dodecylsulfate-polyacrylamide gel electrophoresis. On the basis of the study by Hitchcock and Brown, it was deduced that the top fraction was mainly long O-side chain LPS and the bottom fraction was O-side chain-less LPS. The middle fraction was a mixture of both short O-side chain LPS and O-side chain-less LPS. The antigenic properties and biological activities were not altered in this fractionation procedure. Comparison of the biological activities of the top fraction with those of the bottom fraction revealed that the bottom fraction had higher activity in polyclonal B-cell activation and spleen-swelling effect and that there was no significant difference in adjuvant activity, ability to render macrophages cytotoxic, induction of colony-stimulating factor and the ability to induce the Schwartzmann reaction. It was suggested that O-side chain makes no contribution to the latter biological activities including adjuvant activity of S. minnesota LPS.     

Non-enzymic Browning of Soy Sauce

Ion exchange resins have been used to separate soy sauce into three fractions of distinctly different composition: a cation fraction, a neutral fraction and an anion fraction. Almost all of the constituents responsible for browning were recovered in these three fractions.

Storage experiments show that when the three fractions were stored separately, only the cation fraction darkened considerably. When they were combined and stored, the color of the mixture increased at nearly the same rate as that of the original soy sauce. Neutral sugars are important constituents of the neutral fraction with respect to browning. The browning rate of a sugar-amino acid mixture (simulated soy sauce), was about 10% of soy sauce. The effect of the anion fraction (mainly caused by organic acids) and the ashed cation fraction on the over-all browning of soy sauce is calculated to be 1O~12% and 20%, respectively.

The sum of the contribution rate of the anion fraction, the neutral fraction, the amino acids and the ashed cation fraction in the browning of soy sauce was concluded to be approximately 40%. Compounds responcible for residual part of 60% should be considered to exist in the cation fraction. It was suggested that such compounds have strong reducing power and 0₂-uptaking ability.     

The aging of a brain soluble fraction modifies its effect on the activity of neuronal Na+, K+-ATPase

In previous work we presented evidence showing that a brain soluble fraction was necessary to observe the stimulation of membrane Na+,K+-ATPase activity by catecholamines. Preliminary experiments suggested to us that the soluble fraction by itself was able to modify this enzyme activity. In the present study we have assayed the activity of synaptosomal Na+,K+-ATPase in the presence of a soluble fraction (aqueous supernatant after 100,000 g 30 min) prepared from rat cerebral cortex. The soluble fraction was used at different times after its preparation and different conditions in the incubation period previous to the enzyme assay were tested. It was observed that the enzyme activity increased 70% in the presence of a "0 min" soluble fraction. This effect was not found: a) in the presence of a "30 min" soluble fraction or b) when the membranes plus a "0 min" soluble fraction were incubated for 30 min (15 min at 37 degrees C + 15 min at 0 degree C) before the ATPase assay. In the presence of a "60 min" or "24 h" soluble fraction Na+,K+-ATPase activity was inhibited 50%. Results obtained indicate that Na+,K+-ATPase activity of synaptosomal membranes can be stimulated, inhibited or unchanged, depending on the aging of the soluble fraction.     

Different localization of receptors for omega-conotoxin and nitrendipine in rat brain

The bindings of radioiodinated omega-conotoxin GVIA and [3H]-nitrendipine to subcellular fractions of rat brain were examined. The results indicated that omega-conotoxin binding site was mainly present in the mitochondrial fraction, whereas nitrendipine binding site was rich in the mitochondrial but also present in the post-mitochondrial fraction. Fractionation of the mitochondrial fraction on a sucrose density gradient centrifugation showed that the both binding sites were localized in the heavy synaptosomal fraction. These results strongly suggest that the N- and L-type voltage-sensitive calcium channels have different localizations.     

The metabolism in vivo of 3 alpha, 5 beta-tetrahydroaldosterone in the guinea pig

Analysis of urinary metabolites of [1, 2-3H]-3 alpha, 5 beta-tetrahydroaldosterone was performed in male guinea-pigs. Separation of urinary metabolites was carried out by means of DEAE-Sephadex A-25 column chromatography and four fractions (fraction A to fraction D) were detected. 46-50% of fraction A was extractable with ethyl acetate. When ethyl acetate extract was submitted to reversed phase high pressure liquid chromatography, a number of peaks were present and one of these peaks cochromatographed with 3 alpha, 5 beta-tetrahydroaldosterone. Fraction B, fraction C and fraction D were incubated with enzyme and the free steroid released was identified on the basis of retention time on reversed phase high pressure liquid chromatography. Thus, fraction B contained monoglucosiduronate of 3 alpha, 5 beta-tetrahydroaldosterone, whereas identification of fraction C was not successful. Fraction D was characterized as a monosulphate; 3 beta, 5 beta-tetrahydroaldosterone, 3 alpha, 5 beta-tetrahydroaldosterone and 3 beta, 5 alpha-tetrahydroaldosterone were identified as aglycones. It is probable that 3 beta, 5 alpha-tetrahydroaldosterone is converted from other tetrahydroisomers by intestinal bacteria of the guinea-pig, since mammalian tissues do not contain enzymes that introduce a double bond into ring A.     

Interaction of fatty acid binding protein with microsomes: removal of palmitic acid and retinyl esters.

[14C] palmitic acid or [3H] retinyl esters incorporated in microsomal membranes were removed by a cytosolic fraction enriched in fatty acid binding protein. When mouse liver cytosol was fractionated by 70% ammonium sulphate, a precipitate and a soluble fraction were obtained. The soluble fraction containing the fatty acid binding protein was able to remove from microsomal membranes, [14C] palmitic acid or [3H] retinyl esters, whereas the precipitate fraction had no removal capacity. Retinoid analysis indicated that 70% ammonium sulphate soluble fraction was enriched in endogenous retinyl esters with regard to cytosol or 70% ammonium sulphate precipitate fraction.     

Characterization of membrane-bound spermidine dehydrogenase of Citrobacter freundii.

Spermidine dehydrogenase found in the membrane fraction of Citrobacter freundii IFO 12681 was solubilized with Triton X-100 and further purified to homogeneity. The properties of the membrane enzyme were almost identical to those obtained from the soluble fraction of the organism with respect to molecular and catalytic properties. Thus, binding properties of the enzyme to the bacterial membrane were checked. The ratio of enzyme activity found in the soluble fraction to the membrane fraction was dependent on salt concentration during cell disruption. A hydrophobic interaction was largely involved in anchoring the enzyme to the membrane fraction. Purified spermidine dehydrogenase from the soluble fraction was readily adsorbed into the membrane fraction in the presence of salt. Spermidine dehydrogenase appeared to be a membrane-bound enzyme localized in the cytoplasmic membranes in a manner that makes a partial release of the enzyme possible during mechanical cell disruption. When spermidine oxidation was done with the resting cells of C. freundii, a stoichiometric formation of two reaction products, 1,3-diaminopropane and gamma-aminobutyraldeyde, was observed without any lag time. These facts indicate that the enzyme is localized on the outer surface of the cytoplasmic membranes or in the periplasmic space of the organism.     

Exchange of phospholipids between brain membranes in vitro

1. When unlabelled mitochondria from guinea-pig brain were incubated with a (32)P-labelled microsomal fraction from brain there was a transfer of phospholipid to the mitochondria, which could not be accounted for by an aggregation of microsomes and mitochondria or an exchange with microsomes contaminating the mitochondria. Under similar circumstances there was a transfer of phospholipid from (32)P-labelled mitochondria to microsomes, indicating that the process was one of exchange. 2. The transfer from microsomes was greatly stimulated by a non-dialysable heat-labile macromolecular component in the brain supernatant fraction but not by the concentration of the particulate fractions. 3. Phospholipid-exchange processes occurred most readily between pH7 and 7.5 and were inhibited by the presence of myelin and on the addition of lysophosphatidylcholine. 4. The rates of transfer of individual phospholipids from brain microsomes to mitochondria were similar. 5. (32)P-labelled microsomes could slowly donate phospholipid to the isolated synaptosomal (nerve-ending) fraction but the phospholipids of the myelin fraction did not exchange. 6. Subfractionation of the synaptosomal fraction after [(32)P]phospholipid transfer showed that the mitochondria were most actively labelled during the incubation. All of the isolated individual synaptosomal membranes were capable of acquiring phospholipid on incubation with a (32)P-labelled brain supernatant fraction although a greater percentage was again exchanged by the mitochondrial fraction.     

Molecular weight analysis of soluble antigens from Toxoplasma gondii

Ultrasonicated Toxoplasma gondii (RH strain) tachyzoites were fractionated into a water-soluble and a deoxycholate-soluble fraction. Polyclonal immune mouse serum was prepared by challenging chronically-infected mice with viable RH strain tachyzoites. The parasite fractions were labelled with 125I, and the radio-labelled antigens were precipitated by the immune mouse serum or a monoclonal anti-Toxoplasma antibody (FMC 20), that reacts only in the indirect hemagglutination antibody test. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the immunoprecipitates showed that the water-soluble fraction contained 10 antigenic polypeptides, and the deoxycholate-soluble fraction contained seven antigenic peptides. The FMC 20 reacted against a 98,000-dalton antigen that was present in the water-soluble fraction only.