Complement proteins C5b-9 induce transbilayer migration of membrane phospholipids.

Transbilayer migration of membrane phospholipid arising from membrane insertion of the terminal human complement proteins has been investigated. Asymmetric vesicles containing pyrene-labeled phosphatidylcholine (pyrenePC) concentrated in the inner monolayer were prepared by outer monolayer exchange between pyrenePC-containing large unilamellar vesicles and excess (unlabeled) small unilamellar vesicles, using bovine liver phosphatidylcholine-specific exchange protein. After depletion of pyrenePC from the outer monolayer, the asymmetric large unilamellar vesicles were isolated by gel filtration and exposed to the purified C5b-9 proteins at 37 degrees C. Transbilayer exchange of phospholipid between inner and outer monolayers during C5b-9 assembly was monitored by changes in pyrene excimer and monomer fluorescence. Membrane deposition of the C5b67 complex (by incubation with C5b6 + C7) caused no change in pyrenePC fluorescence. Addition of C8 to the C5b67 vesicles resulted in a dose-dependent decrease in the excimer/monomer ratio. This change was observed both in the presence and absence of complement C9. No change in fluorescence was observed for control vesicles exposed to C8 (in the absence of membrane C5b67), or upon C5b-9 addition to vesicles containing pyrenePC symmetrically distributed between inner and outer monolayers. These data suggest that a transbilayer exchange of phospholipid between inner and outer monolayers is initiated upon C8 binding to C5b67. The fluorescence data were analyzed according to a "random walk" model for excimer formation developed for the case where pyrenePC is asymmetrically distributed between lipid bilayers. Based on this analysis, we estimate that a net transbilayer migration of approximately 1% of total membrane phospholipid is initiated upon C8 binding to C5b67. The potential significance of this transbilayer exchange of membrane phospholipid to the biological activity of the terminal complement proteins is considered.     

Temperature and concentration behavior of anomalous microwave resonances in DNA

We have calculated the expected absorption of microwave radiation in the gigaHertz frequency range by fixed-length DNA polymer molecules dissolved in saline solution. While the effects of counterions and solvent dynamics have been accounted for in detail, the features of the absorption are completely dominated by the interaction between the charged polymer and the so-called first hydration layer, that is, the nearest layer of solvent water molecules not actually bonded to the polymer. The relevant parameters of the interaction are the strength of the water-to-polymer coupling and the average persistence time of the individual water-to-polymer bonds. These are presumably hydrogen bonds to the oxygen atoms of the backbone phosphate structure. Using a given parameterization we can obtain the structured absorption corresponding to compressional wave phonon excitations on the polymer, "organ pipe" modes, such as have been claimed to be seen by Edwards, Davis, Swicord, and Saffer. While further studies have not confirmed these resonances, at some frequency and hydration these modes must become visible because of the high relaxation time measured by Lindsay, the existence of the resonances in relatively dry fibers and films of DNA, and the existence of underdamped modes in the ir spectrum of DNA in solution. We have examined the effects of varying salt concentration and the system temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methyl linoleate ozonide as a substrate for rat glutathione S-transferases: reaction pathway and isoenzyme selectivity

The 9,10-mono-ozonide of methyl linoleate was shown to be a substrate for rat hepatic cytosolic, rat lung cytosolic and rat hepatic microsomal glutathione S-transferases (GST). The activities of lung cytosol and liver microsomes with methyl linoleate ozonide (MLO) were found to be high relative to the activity demonstrated by liver cytosol, as compared with their respective activities towards 1-chloro-2,4-dinitrobenzene (CDNB). Only a slight catalytic activity towards the ozonide was noticed for rat lung microsomes. Isoenzyme 2-2 exhibited the highest specific activity (208 nmol/min/mg) when isoenzymes 1-1, 1-2, 2-2, 3-3, 3-4, 4-4 and 7-7 were compared. This isoenzyme accounts for approx. 25% of cytosolic GST protein in rat lung, while in rat liver it represents approx. 9%. This may partly explain the high activity towards the ozonide noticed for rat lung cytosol. No stable conjugates were formed as products of the reaction of MLO with glutathione; although two glutathione-conjugates were noticed on TLC, they were only formed as intermediate compounds. Coupling of an aldehyde dehydrogenase assay or a glutathione reductase assay to the GST-catalyzed conjugation, demonstrated that oxidized glutathione and aldehydes are formed as the major products in the reaction. To further confirm the formation of aldehydes, the products of the GST-catalyzed reaction were incubated with 2,4-dinitrophenylhydrazine, which resulted in hydrazone formation. In conclusion, the activity of the GST towards the ozonide of methyl linoleate is similar to their peroxidase activity with lipid hydroperoxides as substrates.     

TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp

1-Aminocyclopropane-1-carboxylate synthase was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two-dimensional gel electrophoresis indicates that 1-aminocyclopropane-1-carboxylate synthase activity is associated with a 45-kDa polypeptide, with a pI of 5.8 +/- 0.2. The enzyme is inactivated both by its substrate, S-adenosyl-L-methionine (AdoMet) and by one of its products, 1-aminocyclopropane-1-carboxylate. Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi-industrial scale and 1-aminocyclopropane-1-carboxylate synthase purified. This yielded approximately 150 micrograms enzyme.     

The gene encoding dihydrolipoyl transacetylase from Azotobacter vinelandii. Expression in Escherichia coli and activation and isolation of the protein

The gene encoding the dihydrolipoyl transacetylase (E2) component from Azotobacter vinelandii has been cloned in Escherichia coli. High expression of the gene was found when the cells were grown for more than 14 h. The E2 produced was partially active, varying 10 and 90% in different experiments. By limited proteolysis of the protein it was shown that the catalytic domain was incorrectly folded, caused by formation of intermolecular or intramolecular S-S bridges. The enzyme was fully activated after unfolding in 2.5 M guanidine hydrochloride containing 2 mM dithiothreitol, followed by refolding by dialysis. Active E2 was isolated in a simple three-step procedure. It possessed a specific activity in the same order as that found after isolation of E2 from purified pyruvate dehydrogenase complex from A. vinelandii. Active E2 comprises about 7% of the total soluble cellular protein in the E. coli clone. By genetic manipulation, deletion mutants of E2 were created, one encoding the lipoyl domain and the N-terminal half of the pyruvate-dehydrogenase (E1)- and lipoamide-dehydrogenase (E3)-binding domain, the other encoding the catalytic domain and the C-terminal half of the E1- and E3-binding domain. In E. coli expression of both mutants was observed.     

Association of mRNA and eIF-2 alpha with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

Normal phenotype and slight mental retardation in de novo distal 8p deletion (8pter----8p23.1:)

In this report we present a 9-year-old boy with mental retardation, behavioural problems and terminal deletion of the short arm of chromosome 8(8pter----8p23.1:). In contrast with previously reported patients with larger terminal and interstitial 8p deletions he did not present major phenotypic abnormalities.     

Partial duplication of the short arm of chromosome 2 (dup(2)(p13----p21) associated with mental retardation and an Aarskog-like phenotype

In this report we describe a 3-year-old boy with partial trisomy of the short arm of chromosome 2 due to a de novo tandem duplication 2(dup(2)(p13----p21)). In addition to severe growth retardation and moderate psychomotor delay he presented a dysmorphic syndrome compatible with the clinical diagnostic of Aarskog syndrome.     

O dealkylation and aliphatic and aromatic hydroxylation of 3-methoxy-17 beta-estradiol by Aspergillus alliaceus.

Aspergillus alliaceus UI 315 was examined for its ability to metabolize 3-methoxy-17 beta-estradiol. Preparative-scale incubations with this substrate afforded good yields of 6 beta-hydroxy-17 beta-estradiol, 4-hydroxy-17 beta-estradiol, and 4,6 beta-dihydroxy-17 beta-estradiol, which were identified by high-pressure liquid chromatography, 1H and 13C nuclear magnetic resonance, and high-resolution mass spectrometry.