Linoleic acid hydroperoxide favours hypochlorite- and myeloperoxidase-induced lipid peroxidation

Liposomes composed of soybean phosphatidylcholine were peroxidized using the reagent sodium hypochlorite or the myeloperoxidase-hydrogen peroxide-Cl^- system. Linoleic acid hydroperoxide previously prepared from linoleic acid by means of lipoxidase was incorporated into liposomes. The yield of thiobarbituric acid reactive substances (TBARS) continuously increased with higher amounts of hydroperoxide groups after the initiation of lipid peroxidation by hypochlorous acid producing systems. The accumulation of TBARS was inhibited by scavengers of free radicals such as butylated hydroxytoluene and by the scavengers of hypochlorous acid, taurine and methionine. Lipid peroxidation was also prevented by sodium azide or chloride free medium in the myeloperoxidase-hydrogen peroxide-Cl^- system. Here we show for the first time that the reaction of hypochlorous acid with a biologically relevant hydroperoxide yields free radicals able to cause further oxidation of lipid molecules.     

Subtle re-location of dendritic spine branches containing membrane with fast spiking mechanisms can alter synaptic efficacy

The potential physiological impact of morphological changes in the active dendritic spines, which are believed to be associated with altered synaptic efficacy, was investigated in a computer simulation study using the NEURON package [1]. A compartmental model of a simplified neuron was built, which included 30 complex spines (neck, head, and active zone) and accommodating AMPA-type synaptic inputs with alpha-function conductances. Hodgkin-Huxley type excitable membranes were inserted into the spine heads. It was shown that arranging spines in dense clusters, as opposed to a uniformly random spine distribution, has a negligible effect on the synaptic signal transfer (other model conditions, including synaptic input and spine density, remained unchanged). However, if a proportion (e.g., 3–20%) of the spines partly fuse with their neighbors forming branched spines, this could increase dramatically the cell response to the unchanged synaptic input. Results of this pilot study provide the basis for a more detailed investigation of the relationship between the spine arrangement and synaptic function, considering dual-component synaptic currents and mechanisms controlling ion fluxes in the dendritic compartments.     

Contents of polyamines during vernalization in wheat and the effect of zearalenone

The contents of endogenous free and conjugated polyamines, putrescine (Put) and spermidine (Spd), were determined during 9 week of vernalization (at 5 °C) in winter wheat seedlings cultivated on Murashige and Skoog media without (MS0) and with 2 mg dm⁻³ zearalenone (MSZEN). At the 4^th week of chilling treatment, which is sufficient to induce generative development in 30 % of plants, the marked increase in free and conjugated forms of Put and free Spd were observed. The presence of ZEN in medium significantly accelerated the vernalization. About 20 % of plants treated with ZEN flowered already after 2 weeks and 40 % after 3 weeks of chilling. Significantly higher content of free Put was determined in roots grown on MSZEN compared with MS0 during the first 5 weeks of vernalization with maximum at the 4^th week. After germination, a marked decrease in free Spd content was observed both in plants grown on MS0 and MSZEN. Application of ZEN significantly slowed down the Spd decline in leaves and roots during the first and second week of vernalization. The content of Spd and its conjugates decreased in vernalized plants after 1 week of cultivation at 20 °C.     

Two-dimensional crystallization of bovine rhodopsin

Bovine rhodopsin has been clustered into two-dimensional crystals in highly purified native rod disk membranes and studied with negative staining and transmission electron microscopy. The lattice is P2(1) with dimensions of 8.3 X 7.9 nm and interaxis angles of 86 +/- 3 degrees. 110 images of ordered areas were digitized and aligned with computer-correlation methods to calculate an average image with diffraction to the fourth order. The images were computer-filtered and reconstructed to approx. 2 nm resolution. When crystals appeared they covered 20-40% of the surface of the preparation and, since rhodopsin is at least 95% of the protein, there is no doubt that the crystals were due to rhodopsin. There appear to be two rhodopsin dimers per unit cell. Each rhodopsin molecules takes up about 7.5 nm2 of membrane area and is estimated to be associated with about 12 lipids on each side of the membrane. The membrane area found for bovine rhodopsin supports the rhodopsin origin of rarely seen but more highly ordered two-dimensional crystals found in detergent-treated frog rod membranes (Corless, J.M., McCaslin, D.R. and Scott, B.L. (1982) Proc. Natl. Acad. Sci. USA 79, 1116-1120). Furthermore, the rhodopsin membrane area is close to that of bacteriorhodopsin and is consistent with a seven transmembrane helix structure proposed for rhodopsin (for references see Dratz, E.A. and Hargrave, D.A. (1983) Trends Biochem. Sci. 8, 128-131). Crystallization was accomplished by lowering the pH to 5.5 near the isoelectric point of rhodopsin, raising the salt concentration of 2 M (NH4)2SO4, adding 5% glucose and 0.02% Hibitane (Ayerst), a cationic amphipathic antiseptic that favored crystal growth.     

The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.     

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.     

Purification of human plasma fibronectin by double affinity chromatography on gelatin and heparin-Trisacryl

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.     

Immunohistochemical evaluation of ras oncogene expression in pulmonary and pleural neoplasms

We undertook an immunohistochemical analysis of human bronchopulmonary epithelial neoplasms and pleural mesotheliomas using a monoclonal antibody which recognizes ras oncogene products (p21ras). The monoclonal antibody, RAP-5, recognizes both unaltered and certain mutated p21ras. Formalin fixed and paraffin embedded tissue samples of 187 lung epithelial tumors and 27 pleural mesotheliomas were investigated; normal and bronchiectatic lungs were similarly studied. Normal lung and pleural tissue did not immunostain except for occasional type II pneumocytes. Reactive type II pneumocytes adjacent to carcinomas and bronchiectasis immunostained consistently. Twenty four/34 (71%) squamous carcinomas immunostained. Only 8/50 (16%) adenocarcinomas immunostained focally and weakly whereas 19/24 (79%) bronchioloalveolar carcinomas immunostained. Eleven/18 (61%) large cell carcinomas immunostained with variable intensity. Eleven/13 (85%) carcinoids, 6/7 (85%) well differentiated neuroendocrine carcinomas, and 18/21 (86%) intermediate cell neuroendocrine carcinomas immunostained while none of 20 small cell neuroendocrine carcinomas immunostained. Only a few mesotheliomas were immunostained focally. Two/14 (14%) epithelial type and 1/9 (11%) biphasic type mesotheliomas immunostained weakly; none of 4 spindle cell mesotheliomas immunostained. We conclude that while at least occasional cases of most types of pulmonary epithelial neoplasms express p21ras, the frequency and intensity of the expression are distinctly greater in certain tumor types such as squamous, bronchioloalveolar, and neuroendocrine neoplasm except for the small cell type. Contrary to these lung epithelial neoplasms, most mesotheliomas did not immunostain for p21ras. Whether the enhanced p21ras expression may point to a different mechanism of transformation or may merely reflect differentiation features remains undetermined.     

Interaction of the organic anion 1-aniline 8-naphthalene sulfonate (ANS) with isolated rat hepatocytes

The interaction of ANS with rat hepatocytes in time was studied by fluorescence spectroscopy. The intercept of the first linear portion of the time curve of interaction showed a positive value over all the ANS concentration range employed. This value was maintained after cellular disruption by homogenization. It was affected by ionic strength, pH, and divalent cation in the incubation medium, all conditions affecting the cellular surface. These data suggest that this phenomenon might be a binding of the compound to the hepatocytes surface. Due to the time constant and its disappearance after cellular disruption the other slower component of the curve seems to correspond to a process of translocation across the membrane.     

Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.