Ultrastructural characterization of the sexually dimorphic medial preoptic nucleus of male Japanese quail

The medial preoptic nucleus is a sexually dimorphic structure whose cytoarchitecture, afferent and efferent connections, and functions have been previously described. No detailed ultrastructural study has, however, been perfomed to date. Here we describe the ultrastructural organization of this important preoptic structure of the male quail. Neuronal cell bodies of the medial preoptic nucleus generally show extensive development of protein-synthesis-related organelles (rough endoplasmic reticulum, polysomes), and of secretory structures (Golgi complexes, secretory vesicles, dense bodies). Previous morphometrical studies at the light-microscopical level have demonstrated the presence of a medial and a lateral neuronal population distinguished by the size of their cell bodies (the medial neurons are smaller than the lateral neurons). The present ultrastructural investigation confirms the difference in size, but no difference has been observed in the ultrastructural organization of the neurons. In both the medial and the lateral part, the nucleus is characterized by a large variety of cell bodies, including some that, on the basis of their ultrastructure, can be considered as putative peptidergic neurons. Close contacts are frequently observed between adjacent cell bodies that are normally arranged in clusters. Various types of synaptic endings are also present, suggesting a rich supply of nerve fibers. A few glial cells are scattered within the nucleus. In view of the crucial role of this region in regulating quail sexual behavior, the large heterogeneity of neurons and of afferent nervous fibers suggest that this region might have an important role in the integration of information arriving from different brain regions.     

Leucine transport in Xenopus laevis oocytes: Functional and morphological analysis of different defolliculation procedures

l-leucine uptake in stage V Xenopus laevis oocytes was affected by the specific methods used to remove the follicle cells. In the presence of 100 mM NaCl, l-leucine uptake was reduced by 67.5%±5.7 when defolliculation was performed enzymatically by collagenase treatment, whereas the reduction was 30.5%±6.4 after mechanical defolliculation. The Na⁺-dependent uptake of 0.1 mM l-leucine was 18.6±4.6 pmol oocyte⁻¹ 40 min⁻¹ in folliculated oocytes and 5.6±1.9 in collagenase defolliculated oocytes (means±SE). l-leucine uptake was not affected by the removal of the follicular layer if defolliculation occurred after the transport period; radiolabeled l-leucine is therefore not taken up into a compartment that is removed by the defolliculation process. The different l-leucine uptake rates observed in folliculated and defolliculated oocytes were not due to non-specific l-leucine binding to membranes. l-leucine kinetics showed that the l-leucine V_max and K_m values were lower in oocytes deprived of the follicular layer than in control oocytes enveloped in intact follicular layers. The V_max and K_m values of Na⁺-dependent l-leucine transport, calculated from data obtained the day after defolliculation by collagenase treatment, were: 16±1.5 pmol oocyte⁻¹ 40 min⁻¹ and 57±21 μmol (mean±SD). The Na⁺-activation curve of 0.1 mM l-leucine was hyperbolic in folliculated oocytes and sigmoidal in defolliculated oocytes. The morphological analysis performed in parallel with the transport experiments showed that after defolliculation, the fibers forming the vitelline membrane tended to be arranged in a more regular orthogonal array, and the number of oocyte microvilli was reduced after collagenase treatment. Mechanical defolliculation did not appreciably affect the oocyte microvilli, however this procedure did not completely remove all follicle cells. The damage to collagenase treated oocytes was reversible, and the functional and structural features of most oocytes improved upon subsequent in vitro incubation. The recovery process seemed to involve protein synthesis in view of the increased value of l-leucine V_max, and microscopic observation showing recovery of the microvillar apparatus.     

Membrane-phorbol ester interactions monitored by circular dichroism

The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.     

In vitro stimulation of mouse NK cell activity by phorbol esters: A pathway different from interferon-induced activation

Tumor promoters of the phorbol diester type were tested in vitro for their effect on mouse natural killer (NK) activity. The 12-O-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased the spontaneous cytotoxicity of spleen cells preincubated with this reagent, with a maximal effect at 10–20 ng/ml. Higher concentrations of the weak tumor promoter phorbol-12, 13-dibenzoate (PDB) were required to display the same NK-enhancing property, while the inactive tumor promoter 4-αphorboldidecanoate was ineffective at any concentration. TPA-responding spleen cells displayed the same characteristics as classical NK cells: they were present in nude mice, did not adhere to nylon wool, bore the Thy-1 antigen, and displayed the same target cell specificity as interferon-activated NK cells. A major difference with the interferon-induced NK activation resides in the fact that the TPA-inducible increase in lytic activity does not require RNA and protein synthesis. Our results suggest two different NK activation pathways for IFN and phorbol esters.     

Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways

Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.     

Nuclear protein-kinase activity in perfused rat liver stimulated with dibutyryl-adenosine cyclic 3':5'-monophosphate.

Cytoplasmic and nuclear protein kinase activities from perfused rat liver have been studied in response to dibutyryl-adenosine cyclic 3':5'-monophosphate added at a concentration that stimulates hepatic gluconeogenesis (100 muM). Total nuclear protein kinase, as assayed using a mixed histone fraction as phosphate acceptor, is increased by 5-fold within 8 min of the addition of cyclic nucleotide to the perfusate. In contrast the total cytoplasmic protein kinase activity is decreased to 50% of the control value. The protein substrate specificity of the protein kinase that is present in the nucleus in response to dibutyryl-adenosine cyclic 3':5'-monophosphate stimulation is similar to that of cytoplasmic, adenosine cyclic 3':5'-monophosphate-dependent, protein kinase but is distinct from that of the enzyme(s) present in control nuclei. The predominant species to protein kinase from stimulated nuclei has a sedimentation constant of 3.9 S. This value is identical to that of the catalytic subunit of cytoplasmic adenosine 3':5'-monophosphate-dependent protein kinase. These data suggest that some of the effects of adenosine 3':5'-monophosphate on nuclear events may be mediated through its interaction with the inactive protein kinase holoenzyme in the cytoplasm and the subsequent redistribution of the active catalytic subunits generated by this interaction.     

Isoelectric beads for proteome pre-fractionation. II: experimental evaluation in a multicompartment electrolyzer

Proteome pre-fractionation in multicompartment electrolyzers is proposed here, with substantial modifications as compared to the standard technique. First of all, the classical isoelectric, buffering membranes, delimiting each compartment and acting, in pairs, as isoelectric traps, have been replaced by isoelectric buffering beads, operating on the same principle, but allowing unhindered migration of proteins (lack of sieving properties, contrary to typical continuous membrane barriers). Secondly, the isoelectric beads are not made in the conventional manner, with ionic acrylamide derivative monomers throughout their central core, but are composed of a hard, ceramic core, coated with an amphoteric buffering polymer. This minimizes mass transfer resistance of proteins that are transiently adsorbed onto the beads. As a result, significantly reduced separation times, of the order of ca. 3 h, are required for developing steady-state patterns, as compared to the lengthy times (overnight and much longer) in conventional multicompartment electrolyzers operating with isoelectric membranes. Examples of separation of standard marker proteins, as well as entire Escherichia coli lysates and human serum proteins, are given. The obtained fractions are analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by surface enhanced laser desorption/ionization mass spectrometry.