Reactivity of HDL subfractions towards lecithin-cholesterol acyltransferase. Modulation by their content in free cholesterol

(1) Human HDL2 (d 1.070-1.125) and HDL3 (d 1.125-1.21) labelled with unesterified [14C]cholesterol, were incubated with a source of lecithin-cholesterol acyltransferase. For optimal activity, the reaction required the addition of albumin in excess, at least 3-times greater than the concentration of HDL-free cholesterol. Under such conditions, the reaction appeared saturable. HDL3 was found the most efficient substrate and the Vmax values expressed for 1.5 IU LCAT/ml and with an albumin/free cholesterol ratio of 3, were 8.3 nmol free cholesterol esterified/ml per h and 4.1 nmol/ml per h for HDL3 and HDL2, respectively. (2) HDL3 were modified in the presence of VLDL by inducing triacylglycerol lipolysis with a semipurified lipoprotein lipase from bovine milk. The newly formed HDL had gained free cholesterol and phospholipids, so that about 50% of these modified HDL, referred to as light-LIP-HDL3, were reisolated in the HDL2 density range. Light-LIP-HDL3 were enriched mostly in free cholesterol (+ 160%) and in phospholipid (+ 40%). Their reactivity towards LCAT was half-reduced compared to parent HDL3, which correlated well with a decrease in their phospholipid/free cholesterol molar ratio. Moreover, HDL3 artificially enriched in free cholesterol and exhibiting a comparable PL/FC behaved like lipolysis-modified HDL in their reactivity towards LCAT. (3) HDL3 were also modified by co-incubation with VLDL (post-VLDL-HDL3), or with VLDL and a source of lipid transfer protein (CET-HDL3). The latter treatment greatly affected the lipid composition of the core particle (-25% esterified cholesterol, +190% TG). In both cases, the moderate decreasing LCAT reactivity observed could be related to the phospholipid/free cholesterol ratio. Thus, like in artificial substrates, the lipid composition of the HDL surface may control the rate of LCAT-mediated cholesterol esterification.     

Biochemical dissection of the role of the one-kilodalton carboxyl-terminal moiety of tubulin in its assembly into microtubules

The 4-kDa C-terminal domain of both tubulin subunits plays a major role in the regulation of microtubule assembly [Serrano et al. (1984) Biochemistry 23, 4675]. Controlled proteolysis of tubulin with subtilisin produces the selective cleavage of this 4-kDa moiety from alpha- and beta-tubulin with a concomitant enhancement of the assembly. Here we show that gradual removal of the last six to eight amino acid residues of the C-terminal region of alpha and beta subunits by an exopeptidase, carboxypeptidase Y, produces a modified protein (C-tubulin) without relieving the modulatory effect of the C-terminal domain and the usual need of MAPs for microtubule assembly. Actually, treatment with this proteolytic enzyme did not change tubulin assembly as promoted by either MAP-2, taxol, MgCl2, dimethyl sulfoxide, or glycerol. The critical concentration for the assembly of C-tubulin remained the same as that for the unmodified tubulin control. Microtubule-associated proteins MAP-2 and tau incorporated into C-tubulin polymers. Clearly, pure C-tubulin did not assemble in the absence of MAPs or without addition of assembly-promoting compounds. However, proteolysis with the exopeptidase induced changes in tubulin conformation as assessed by biophysical methods and double-limited proteolysis. The cleavage with subtilisin after carboxypeptidase digestion did not result in enhancement of the assembly to the levels observed after the treatment of native tubulin with subtilisin. Interestingly, Ca2+ ions affected neither C-tubulin assembly nor depolymerized microtubules assembled from C-tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ uptake or the amount of Ca²⁺ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca²⁺ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) vesamicol inhibited both the fall in membrane potential and the elevated Ca²⁺ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na⁺ channels at this concentration. Vesamicol, in lower concentration (20 M) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [¹⁴C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [¹⁴C]choline. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

Fluorescent labeling of nitrocellulose-bound proteins at the nanogram level without changes in immunoreactivity

Proteins blotted on nitrocellulose were stained with either 5-dimethylamino-1-naphthalene-sulfonylchloride (dansyl chloride) or fluorescein isothiocyanate. In both cases the staining procedure can be completed in less than 30 min. The sensitivity for detecting fluorescent-labeled proteins on nitrocellulose was 0.5 ng using a dot test. This was accomplished by transparentizing the nitrocellulose with either immersion oil or toluene. Dansylated proteins were successfully utilized for optimizing the electroblotting procedure. In the presence of 0.2% sodium dodecyl sulfate and 20% methanol the distribution of proteins on the nitrocellulose was an exact replica of the protein pattern seen in the polyacrylamide gel. The fluorescent labeling did not affect the antigenic properties of proteins allowing the subsequent probing with antisera. For this procedure, only one set of samples is needed to obtain accurate photographic records of the gel, the nitrocellulose blot, and the probed blot.     

The expression of the sIgD isotype in wild-derived mice

IgD and IgM are concomitantly expressed on the surface of most mouse B lymphocytes and both molecules serve as receptor for antigen. In this communication we report that in contrast to IgM, which is expressed in a constant manner on the surface of spleen B lymphocytes of different laboratory and wild-derived mice, IgD expression is variable among the spleen cells of wild-derived mice. SPE, SEI, and SFM mice belonging to the Mus 3 subgroup show a fluorescence profile characterized by a marked diminution in the population of B lymphocytes expressing the IgD isotype; in addition, these cells have a low sIgD density on their membranes. These findings were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the iodinated membrane proteins. Polyclonal in vitro activation with lipopolysaccharide increases the frequency of surface IgD (sIgD)-bearing spleen cells and sIgD density in the SPE strain but decreases both the frequency and the density of IgD bearing cells in the BALB/c strain. This result suggests that delta gene expression is regulated differently in SPE and BALB/c mice. In addition, genetic analysis of sIgD expression in (BALB/c X SPE)F1 hybrids suggests that the proportion of sIgD-bearing cells and sIgD density are independently regulated.     

Polypeptide composition of rat sperm outer dense fibers. A simple procedure to isolate the fibrillar complex

Treatment of caput or cauda epididymal rat sperm with a low concentration (0.05%) of the cationic detergent cetyltrimethylammonium bromide and 30 mM 2-mercaptoethanol solubilized most of the sperm structures except for the sperm head and the outer dense fiber-connecting piece complex. The latter were purified, and about 10% of these complexes are formed by nine fibers attached to the connecting piece. Of these fibers, two are shorter than the other seven and presumably correspond to fibers 3 and 8 (Fawcett, D.W. (1975) Dev. Biol. 44, 394-436). Electron microscopy confirmed the purity of the isolated outer dense fibers and revealed their characteristic irregular cross-sectional shape. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed six major polypeptides (Mr = 87,000, 30,400, 26,000, 18,400, 13,000, and 11,500) with a high content of serine, aspartic and glutamic acids, proline, cysteine, leucine, and tyrosine. Furthermore, several lines of evidence indicate a close structural relationship between the components of 30,400 and 26,000 Da. The six major components of the fibers are phosphorylated at serine residues. These results indicate that the major components of rat sperm outer dense fibers are a unique family of phosphoproteins.     

Dietary docosahexaenoic acid is retroconverted in man to eicosapentaenoic acid, which can be quickly transformed to prostaglandin I3

In a 24 h kinetic study docosahexaenoic acid (DCHA, C22:6n-3) or eicosapentaenoic acid (EPA, C20:5n-3) were given in a single dose to healthy male volunteers. PGI₃-M, the main urinary metabolite of prostaglandin I₃ was below the detection limit in the control periods, but was excreted already in the first 4 h after ingestion of DCHA or EPA and decreased thereafter. Excretion of PGI₂-M did not change significantly. In a second dietary trial DCHA and EPA were given cross-over to 7 healthy male volunteers for 6 days. PGI₃-M was formed after DCHA and EPA in amounts of 35 and 20 % of PGI₂-M and showed a considerable interindividual variation. The structure of PGI₃-M was verified by independant biochemical synthesis. Our data indicate that dietary DCHA is retroconverted to EPA in man, which is quickly transformed - like dietary EPA itself - to prostaglandin I₃. DCHA may therefore serve as a precursor fatty acid for EPA and its cyclooxygenated and lipoxygenated products.     

Microbiological dehydrogenation of polyfunctional Δ5-3β-acetoxy steroids by Corynebacterium mediolanum strain B-964

Summary A high-yield microbiological transformation of polyfunctional ⁵-3-acetoxy steroids, containing an additional ring E, by Corynebacterium mediolanum strain B-964 was carried out, resulting in the corresponding ⁴-3-ketones. It was shown that the type of transformation and the yield of the reaction depend on the degree of saturation of the ring E and on the position of the oxygen-containing substituents in it.     

Comparison of the ribosomal RNA genes in four closely relatedCucurbitaceae

Cucurbitaceae are characterized by a high copy number for nuclear ribosomal RNA genes. We have investigated the genomic ribosomal DNA (rDNA) of four closely related species of this family with respect to structure, length heterogeneity, and evolution. InCucumis melo (melon) there are two main length variants of rDNA repeats with 10.7 and 10.55kb.Cucumis sativus (cucumber) shows at least three repeat types with 11.5, 10.5, and 10.2kb.Cucurbita pepo (zucchini) has two different repeat types with 10.0 and 9.3kb. There are also two different repeat types inCucurbita maxima (pumpkin) of about 11.2 and 10.5kb. Restriction enzyme mapping of the genomic rDNA of these four plants and of cloned repeats ofC. sativus shows further heterogeneities which are due to methylation or point mutations. By comparison of the restriction enzyme maps it was possible to trace some evolutionary events in the family ofCucurbitaceae. Some aspects of regulation and function of the middle repetitive rRNA genes (here between 2000 and 10000 copies) are discussed.