Nucleolar structures in chromosome and SC preparations from human oocytes at first meiotic prophase

Summary We describe a comparative study of the behavior of nucleolar structures and their relationship with nucleolar chromosomes and synaptonemal complexes at first meiotic prophase of human oocytes in an attempt to elucidate the nature of this cellular organization and to learn more about maternal nondisjunction. The number of main nucleoli varies along the different stages of prophase I and is usually low. It shows an increase from leptotene to pachytene and a decrease from pachytene to diplotene related to a decrease and an increase of main nucleoli volume, respectively. The methodology employed has enabled us to analyze in detail dark bodies, round bodies, dense bodies, and main nucleoli in chromosome or synaptonemal complex spreads. The relationship between nucleolar chromosomes or synaptonemal complexes and the nucleoli implies the existence, in a very reduced space, of chromosomal regions that contain homologous sequences and that are often unpaired. This situation may facilitate the production of heterologous pairing and chromosomal exchanges between nonhomologous chromosomes and finally result in aneuploidy. THus, the situation explained above together with the differences between the oocyte and spermatocyte NOR cycles could be one of the reasons for the higher incidence of aneuploidies of maternal origin at meiosis I.     

Le collagène du byssus de Mytilus edulis L.

Résumé L'autoradiographie révèle, au niveau du pied, une incorporation massive et sélective de la ³H-Proline dans la glande blanche de Mytilus edulis. Cette étude a permis de suivre le processus qui mène de la synthèse de la sécrétion dans la partie basale des cellules jusqu'a son émission dans le sillon pédieux où elle participe à la formation du filament. La collagénase détruit la presque totalité du marquage, attestant ainsi la nature collagénique du produit sécrété. Les autres glandes pédieuses ainsi que la glande du byssus proprement dite, située à la base du pied, montrent une incorporation très faible, sans commune mesure avec celle de la glande blanche. Ceci démontre de façon définitive que le collagène présent dans le filament prend naissance dans cette glande et justifie la dénomination de glande du collagène. Des contrôles réalisés dans différentes régions (bords du manteau, manteau, branchies) montrent que l'injection du précurseur dans le bord palléal constitue une méthode satisfaisante pour marquer de façon relativement rapide et différentielle le collagène de la glande.

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The collagen of the byssus in Mytilus edulis L.II. Autoradiographic study on the incorporation of ³H-Proline

Summary Autoradiographic studies reveal a strong specific incorporation of ³H-Proline in the white gland in the foot of Mytilus edulis. The author could trace the radioactive secretory product from its synthesis in the basal part of the cells down to its outflow into the pedial groove where it takes part in the formation of the filament. Purified collagenase takes out radioactivity from the sections. This observation confirms the collagenous nature of the secretion.The other foot-glands as well as the main byssus gland located at the base of the foot show but a very weak labelling not comparable with that of the white gland. This clearly evidences that the collagen occuring in the filament originates from the latter. The white gland may be properly called: collagen gland.Control sections through different parts of the body (mantle-edge, mantle, gills) confirm that our injection technique of the precursor into the palleal margin is a suitable method for a rather quick and specific labelling of the glandular collagen.

Cette note fait partie d'un travail pour l'obtention d'une thèse de doctorat.     

Interleukin-1 and osteoarthritis

Il-1, a multifunctional monokine, can stimulate both synoviocytes and articular chondrocytes to release neutral proteases and prostaglandin E2. It is also capable of promoting bone resorption. Therefore, this molecule (or family of molecules) is likely to play an important role in the mechanism of articular cartilage destruction that occurs in degenerative arthropathies. The synovial tissue itself can produce Il-1 (Catabolin) in some conditions, such as a slight traumatism, so that the presence of local inflammation is not necessary for "Il-1-cartilage" interaction to occur. Fundamental macromolecules of cartilage (collagens, proteoglycans) exert a stimulatory effect on Il-1 production, either as such or in the form of immune complexes. Some activated complement fractions (C3a and C5a) may also be actively involved. Studies on the mechanisms which regulate Il-1 synthesis and release, as well as investigations on the response of target cells to Il-1, are presently fascinating goals that could lead to new strategies in therapeutic research.     

Interleukin-1 inhibits the synthesis of collagen by fibroblasts

Human dermal fibroblasts, exposed to human or porcine Interleukin-1, responded by an inhibition of collagen synthesis in a dose dependent manner. Incubation with Il-1 for more than 8 h was required to see an appreciable effect. The phenomenon was not dependent on the presence of serum in the culture medium. Since a stimulation of prostaglandin E2 secretion was also observed in presence of Il-1, we investigated the eventual role of arachidonic acid metabolites in the phenomenon. Inhibitors interfering with arachidonate metabolism, namely indomethacin, acetyl salicylic acid, BW 755 C and NDGA had no influence on the inhibition of collagen synthesis caused by Il-1. These data suggest that both cyclooxygenase and lipoxygenase derived metabolites of arachidonic acid are unlikely to play a role in the mechanism.     

Biochemical and Immunological Properties of the Mouse Brain Enolases Purified by a Simple Method

Abstract: A simple and rapid purification method is presented for the two mouse cerebral isozymes of enolase (EC 4.2.1.11), E₁ and E₃. The purity of the preparations was ascertained by electrophoresis under two different conditions. The biochemical and immunological properties of E₁ and E₃ were compared. The molecular weight of the cerebral enolases was analysed by column chromatography on Sephadex G 150 and by electrophoresis in the presence of SDS. Both E₁ and E₃ are homodimers with a subunit of molecular weight of 50,000. The procedure also yields a semi-purified fraction of E₂. Conditions of in vitro formation of E₂ from pure or semi-purified fractions of E₁ and E₃ show that it is likely to be a real hybrid, rather than an aggregate and that it is probably not an artefact formed during the purification. The K_m values (K_m= 3–4·10^?5 M) for the substrate are not significantly different amongst the three forms. However, E₁ and E₂ but not E₃ are inhibited by excess substrate. Antisera against E₁ and E₃ have been obtained from rabbit and goat, respectively. Antibodies against each protein do not show any cross-reactivity with each other. There is, however, a broad species cross-reactivity, showing conservation of each enolase form during evolution. Both anti-E₁ and anti-E₃ sera react with the E₂ enolase fraction, in agreement with its hybrid structure. Anti-E₃ serum does not react with extracts of other tested organs. Brain enolase 1 resembles liver enolase in its biochemical and immunological properties. A slight cross-reactivity of anti-E₁ serum with muscle extracts is observed. Heterogeneity of brain enolase 1 is observed by both biochemical and immunological methods; the nature of this heterogeneity is discussed.     

Induction of interleukin-1 beta production in human dermal fibroblasts by interleukin-1 alpha and tumor necrosis factor-alpha. Involvement of protein kinase-dependent and adenylate cyclase-dependent regulatory pathways.

It has previously been demonstrated that interleukin-1 (IL-1) is expressed in a variety of fibroblast cell lines. In this study, we investigated the mechanisms involved in the regulation of IL-1 beta production by cultured human dermal fibroblasts. We have shown that IL-1 beta is constitutively expressed as a cell-associated form, with no soluble form detectable in control cell or in stimulated cell supernatants. IL-1 alpha and tumor necrosis factor-alpha (TNF-alpha) exerted a dose-dependent stimulation on the production of the cell-associated IL-1 beta, as estimated using a specific enzyme linked immunosorbent assay (ELISA). As expected, this effect was accompanied by a huge release of prostaglandin E2 (PGE2) and a transient rise in intracellular cyclic AMP. Furthermore, IL-1 beta production was elevated to a lesser extent by the addition of increasing concentrations of the protein kinase C activator phorbol myristate acetate or by low concentration (0.001 microgram/ml) of PGE2. In contrast, higher concentrations (0.1 and 1 micrograms/ml) of PGE2, as well as exogenous dibutyryl-cyclic AMP, were clearly inhibitory. H7, an inhibitor of protein kinases also reduced the stimulatory effect of IL-1 alpha and TNF-alpha. Together with the results obtained with phorbol myristate acetate, these data suggest that protein kinase C may play a role in the upregulation of IL-1 beta expression in normal skin fibroblasts. The addition of indomethacin not only suppressed prostaglandin synthesis, but also dramatically reduced cyclic AMP formation, probably because the PGE2-induced stimulation of adenylate cyclase was abolished. This resulted in a strong potentiation of the stimulatory effect of IL-1 alpha and TNF-alpha, supporting the role of both the cyclooxygenase and adenylate cyclase pathways in the endogenous downregulation of IL-1 beta induction by the two cytokines studied.     

Retrograde axonal transport after radioactive serotonin injections into the olfactory bulb: a biochemical analysis of transported radioactive material

A biochemical analysis of radioactive compounds was performed in the olfactory bulb (OB) and raphe dorsalis (RD) after injection of radioactive [³H] or [¹⁴C]serotonin (5-HT ranging from 10^?2 M to 10^?7 M) into the OB of rats treated or not with a monoamine-oxidase inhibitor (MAOI).In the OB of untreated rats, radioactivity was associated with precipitated protein and soluble perchloric acid (PCA) fractions. High performance liquid chromatography (HPLC) analysis of the PCA-supernatant gave 4 radioactive peaks: one associated with endogenous 5-HT, another with endogenous 5-hydroxyindole acetic acid (5-HIAA) and two without any relationship with endogenous hydroxyindoles: a ‘5-HT derivative A’ and a ‘5-HT derivative B’. The presence of these ‘5-HT derivatives’ was significantly reduced after treatment with 5,6-dihydroxytryptamine.In the RD, radioactivity was associated with the protein fraction and with ‘5-HT derivative A’. The kinetic analysis (from 30 min to 46 h) of the ‘5-HT derivative A’ was characterized by a disappearance in the OB and an accumulation in the RD corresponding to a rate of migration in a range of 0.7 to 2 mm/h. This compound was absent or negligible in other non-serotoninergic neurons (such as the Locus Coeruleus, Amygdala and Cortex piriformis). No clear evidence for retrograde transport of radioactive 5-hydroxytryptophan (5-HTP) or 5-HIAA was found.At lower concentration of 5-HT injected into the OB, the half lives and the times of maximal accumulation for 5-HIAA, ‘5-HT derivative A’ and ‘5-HT derivative B’ were increased. The specific activity of 5-HT and 5-HIAA was also increased.The selective radioactive accumulation in the cell bodies of RD neurons after injection of radioactive 5-HT into the OB is discussed as resulting from a selectivity in (a) the uptake by 5-HT nerve terminals; (b) the metabolism of 5-HT into ‘5-HT derivative A’ in the OB; (c) the retrograde axonal transport of ‘5-HT derivative A’. This ‘5-HT derivative A’ could represent a messenger between nerve terminals and cell bodies and could be involved in homeostatic mechanisms that maintain cellular dynamics.When a MAOI was used, ‘5-HT-derivative A’ and [³H]5-HT were found in the OB and also in the RD cell bodies, and to a lesser extent, in the non-serotoninergic cell bodies. These results indicate that MAO inhibition produces a relative non-selectivity in the ‘uptake-metabolism and retrograde axonal transport’ systems.     

In vivo measurements by differential pulse voltammetry of extra-cellular 5-hydroxyindoleacetic acid in the rat brain

The use of differential pulse voltammetry, performed with electrochemically treated carbon fiber electrodes, enables us to detect in vitro or in vivo, in the striatum of anaesthetized rats, an oxidation peak (3) at a potential of + 300 mV. Electrolytic or 5-7-dihydroxytryptamine lesions of the medial forebrain bundle are followed by a decrease of 59 and 62% respectively of this peak. Biochemical measurements are significantly correlated with the measured peak (3) and decreases. Thus, peak (3) increases obtained after injection of L-tryptophan and/or Reserpine, as well as the decreases observed after injection of Clorgyline or 3-hydroxybenzylhydrazine, confirm that peak (3) is dependent upon 5-hydroxyindoleacetic acid concentration. The detection of a peak (3) in the cerebrospinal fluid and its increase after injection of Probenecid reinforce this conclusion.     

Functional recuperation of the serotoninergic innervation in the rat locus coeruleus

1. 5,6-dihydroxytryptamine (5,6-DHT) or a lesion of the raphe centralis superior (RCS) cause significant decreases in the serotonin (5-HT) content and significant increases in the tyrosine hydroxylase activity in the locus coeruleus (LC) of the rat. This suggests that noradrenaline (NA) synthesis is controlled by serotonin-containing neurons in the raphe system via their terminals in the LC. 2. Radioautography after intraventricular infusion of tritiated serotonin (3H-5-HT) and biochemical determinations of endogenous 5-HT content showed an almost complete disappearance of serotoninergic axonal varicosities and content in the LC region 10-15 days after intraventricular administration of 75 micrograms of 5,6-DHT. Two to 4 months after neurotoxin administration, 5-HT fibers had regrown in the LC but, contrary to the normal innervation pattern, the majority of them invaded the medial most portion of the nucleus and the adjacent subependymal region. The LC region regained almost all of its endogenous 5-HT content in the same time period. 3. Functional recuperation of these 5-HT fibers was demonstrated by the fact that the RCS had, after regeneration, the same functional control on NA synthesis as in the normal animal.     

Developmental studies with the 14-3-2 antigen and the neuron specific enolase (NSE) associated activities—I

We have compared the rodent developmental pattern of the 14-3-2 antigen estimated by a microcomplement fixation technique with that of the cerebral enolases. Chromatographic separation of enolase isozymes on microcolumns demonstrates that the embryonic neuron specific enolase is firstly and mostly represented by the αγ isozyme. The most important increase in 14-3-2 antigen and γγ enolase occurs between post-natal days 7th and 15th. By post-natal day 30, adult levels have been reached. An interesting observation is—during embryonic development—the decrease in the specific activity of the cerebral enolase isozyme αα. This could be explained by the replacement—in neuroblasts—of αα enolase by neuron specific enolase. A comparison between 14-3-2 antigen and neuron specific enolase (γγ) purified by completely different methods is presented. The 14-3-2 antigen exhibits an enolase specific activity comparable to that of purified enzyme and has the same electrophoretic mobility. Antibodies raised against either antigen have an identical specificity. Pre and post-natal developmental pattern in rodent brains are similar for both proteins. Thus neuron specific 14-3-2 antigen is identical to neuron specific enolase.Thus we have precisely described the ontogenic transition between the three cerebral enolase isozymes at the tissue level. This study is completed by the analysis of these transitions at the neuronal cell level, using homogenous cell lines (Part II of this paper).