In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick-translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling.     

A search for an 'ouabain-like' substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na+ + K+)-ATPase activity, the specific binding of [3H]ouabain to purified (Na+ + K+)-ATPase and 86Rb+ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous 'ouabain-like' activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.     

Estrogen and androgen production by purified Leydig cells of mature boars

Purified Leydig cells were obtained from testes of mature male pigs by collagenase treatment and mechanical dispersion, followed by Percoll (0-90%) density gradient centrifugation. The cells recovered at 40-45% Percoll were applied to a second gradient of 15 ml of Percoll (10-60%) to yield three bands, one major and two lesser in numbers of cells. Incubations were then made with 0.25-1.0 X 10(6) cells at 34 degrees C for 3 h in 95% O2: 5% CO2, with or without human chorionic gonadotrophin (hCG) added to the medium. Steroid concentration was determined by radioimmunoassays. The steroids measured in the media were testosterone, dehydroepiandrosterone sulfate (DHAS) and estrone sulfate (E1S). Lesser amounts of dehydroepiandrosterone (DHA) and estrone (E1) were found. Stimulation by hCG led to an increase in apparent steroid production for all steroids, including estrogens, with the greatest quantities seen with DHAS (greater than 200 ng/1 X 10(6) cells/3 h). Cells in the major band gave the best response. These results show that Leydig cells are a significant site of estrogen production in the boar testis and that this organ is a source of an abundant supply of such cells.     

Selection of Wine Yeasts for Growth and Fermentation in the Presence of Ethanol and Sucrose

To optimize the conversion of carbohydrates to ethanol, strains of several Saccharomyces species were examined for the ability to grow and ferment in a range of sucrose and ethanol concentrations. A total of 632 wine yeasts, most of them isolated from wineries in Andalusia and Extremadura, southwestern Spain, were subjected to screening and selection. Growth and fermentative capacity in different ethanol and sucrose concentrations varied from one strain to another. There was no correlation between growth and fermentative capacity. The best 35 strains grew in 15% ethanol and fermented in 18% ethanol. Ethanol accumulated, although at a reduced rate, after the cells stopped growing. Most yeast strains were highly fermentative in 50% sucrose. Some of them effectively utilized the carbohydrates of the culture, yielding final ethanol concentrations of > 14%. Of the 35 selected strains, 16 were promising for genetic analysis and breeding because of their capacity to sporulate. These strains were homothallic, and their spores were viable. The meiotic products analyzed so far were also homothallic.     

Effects of Electrical Stimulation on Molecular Forms of Butyrylcholinesterase in Denervated Fast and Slow Latissimus Dorsi Muscles of Newly Hatched Chicken

The effects of denervation and direct electrical stimulation upon the activity and the molecular form distribution of butyrylcholinesterase (BuChE) were studied in fast-twitch posterior latissimus dorsi (PLD) and in slow-tonic anterior latissimus dorsi (ALD) muscles of newly hatched chicken. In PLD muscle, denervation performed at day 2 substantially reduced the rate of rapid decrease of BuChE specific activity which takes place during normal development, whereas in the case of ALD muscle little change was observed. Moreover, the asymmetric forms which were dramatically reduced in denervated PLD muscle were virtually absent in denervated ALD muscle at day 14. Denervated PLD and ALD muscles were stimulated from day 4 to day 14 of age. Two patterns of stimulation were applied, either 5-Hz frequency (slow rhythm) or 40-Hz frequency (fast rhythm). Both patterns of stimulation provided the same number of impulses per day (about 61,000). In PLD muscle, electrical stimulation almost totally prevented the postdenervation loss in asymmetric forms and led to a decrease in BuChE specific activity. In ALD muscle, electrical stimulation partially prevented the asymmetric form loss which occurs after denervation. This study emphasizes the role of evoked muscle activity in the regulation of BuChE asymmetric forms in the fast PLD muscle and the differential response of denervated slow and fast muscles to electrical stimulation.     

Characterization of rabbit lactate dehydrogenase-M and lactate dehydrogenase-H cDNAs. Control of lactate dehydrogenase expression in rabbit muscle

Two cDNA clones were isolated, one corresponding to the mRNA coding for lactate dehydrogenase-M (LDH-M), the other to the mRNA coding for lactate dehydrogenase-H (LDH-H). The cDNA inserts consist of the entire open reading frame for LDH-M and a partial sequence, from amino acid 117 to 332, for LDH-H. Using these two clones as probes we demonstrate that: (a) the abundance of mRNA is muscle-type dependent; (b) the ratio M/H subunit for protein and mRNA is well related in the muscles studied; and (c) the M + H mRNA level is not relative to the total LDH activity.     

Proton and carbon-13 nuclear magnetic resonance spectroscopy of diastereoisomeric 3- and 17 beta-tetrahydropyranyl ether derivatives of estrone and estradiol

Protection of 3- and 17 beta-hydroxyl groups of estrone and estradiol as tetrahydropyranyl ether derivatives led to mixtures of 2'(R)- and 2'(S)-diastereoisomers which were separated by crystallization (3-tetrahydropyranyl ethers), or by thin-layer chromatography (17-tetrahydropyranyl ethers), and characterized by 1H and 13C nuclear magnetic resonance (NMR). Assignments for NMR signals of estradiol 3,17 beta-ditetrahydropyranyl ether were facilitated by comparison with those of its 15 zeta, 16 zeta-dideuterio analog and by 2D 1H-13C heteroshift correlation experiments. Diastereoisomers of 3-tetrahydropyranyl ether derivatives could be identified through the 13C NMR doublet signals of the anomeric C-2' and the aromatic C-4 carbon atoms in CDCl3. Diastereoisomers of 17-tetrahydropyranyl ether derivatives were recognized from characteristic modifications of 1H NMR signals of H-2', H-6', H-1, H-17, and 18-CH3 protons as well as from the 13C NMR doublet signals corresponding to C-2', C-4', C-6', C-12, C-13, C-16, and C-17 carbon atoms. Low-temperature experiments showed a splitting of the C-2', C-6', and C-17 13C NMR signals of each of the two 17-tetrahydropyranyl ether isomers. The downfield signal (equatorial conformer) of the three resulting doublets was more intense for the 17-tetrahydropyranyl ether 2'(S)-isomer, whereas the upfield signal (axial conformer) was more intense for the 2'(R)-isomer.