Relationship between posttranslational modification of transaldolase and catalase deficiency in UV-sensitive repair-deficient xeroderma pigmentosum fibroblasts and SV40-transformed human cells.

Xeroderma Pigmentosum (XP) is a rare recessively inherited human disease associated with a hypersensitivity to ultraviolet radiation. The ultraviolet component of sunlight can initiate and promote the formation of cutaneous tumors as seen in the skin cancer-prone XP patients. Previously, we have found that the low activity of the NADPH-dependent antioxydant enzyme, catalase, which we have observed in XP diploid fibroblasts and SV40-tranformed cells, could be restored by the addition of NADPH. Here we have analyzed transaldolase, which regulates NADPH levels produced by the pentose phosphate pathway in order to examine how it influences the catalase activity regulated in XP and SV40-transformed cells. We find that transaldolase activity is high in XP and SV40-transformed human fibroblasts, whereas transaldolase transcription is unchanged, suggesting that modification of transaldolase activity is due to a posttranslational modification of the protein. Two-dimensional electrophoresis analysis has allowed us to identify a complex set of transaldolase isoforms and to postulate that the phosphorylation of specific isoforms could be correlated with the different enzymatic activities seen. Our results show that high transaldolase activity corresponds to a low catalase activity in SV40-transformed cells and in fibroblasts from XP patients who have a high predisposition to develop skin cancer.

Neuropeptide-induced inhibition of steroidogenesis in crab molting glands: involvement of cGMP-dependent protein kinase.

In crustaceans, ecdysteroid production by the molting glands (Y-organs) is negatively regulated by a neuropeptide, molt-inhibiting hormone (MIH). The involvement of cyclic nucleotide-dependent kinases in the mechanism of action of this neuropeptide was investigated with regard to the steroidogenic activity of Carcinus maenas Y-organs. Regardless of the activity level, the major phosphotransferase activity measured in cytosolic fraction was cGMP-dependent, indicating a relatively high cytosolic concentration of cGMP-kinase in these cells. Phosphotransferase activity was nearly twofold higher in the intermolt (low steroidogenic activity) than in premolt (high steroidogenic activity) animals. In vitro incubation of premolt Y-organs with MIH for 1 hr increased by 3.7-fold the cGMP-kinase activity ratio (-cGMP/ +cGMP). Numerous endogenous protein substrates were predominantly phosphorylated in a cGMP-dependent manner in cytosolic, particulate, and membrane fractions. Similar phosphoprotein patterns were observed in both molting stages. By contrast, cAMP-kinase activity, which was low in intermolt Y-organs, increased significantly in the active steroidogenic premolt Y-organs. The increase in cAMP-kinase activity was accompanied by a cAMP-dependent phosphorylation of several specific endogenous proteins. Taken together these results strongly suggest that activation of cGMP-kinase and subsequent phosphorylation of an endogenous protein(s) may be responsible, at least in part, for the MIH-induced inhibition of steroidogenesis. By contrast, it is most unlikely that cAMP-kinase is involved in these processes.

A transaldolase : An enzyme implicated in crab steroidogenesis.

In arthropods, development is controlled by cholesterol-derived steroid hormones: the ecdysteroids. In vertebrates and insects, steroidogenesis is positively regulated and this is mediated by cAMP. In crustaceans, ecdysteroid biosynthesis by steroidogenic organs (Y-organs) is negatively regulated by a neuropeptide, the Molt Inhibiting Hormone (MIH). This neuropeptide-induced inhibition occurs via cyclic nucleotides and depends on protein synthesis. In the present work, we provide evidence that a major 36.2-kDa cytosolic protein (P36; pl: 6.8) from crab Y-organs is positively correlated with steroidogenic activity. On the basis of its amino acid sequence, P36 could be related to transaldolase, an enzyme of the pentose phosphate pathway which generates NADPH. In Y-organs, the enzymatic activity ofCarcinus transaldolase increases with steroidogenic activity, and MIH treatment decreases both synthesis and activity of transaldolase. Various transaldolases have been characterized in very distantly related groups, namely bacteria, yeasts, and humans. These enzymes are highly conserved and present strong structural homologies, interestingly the crab transaldolase is closest to that enzyme characterized in human cells.

Involvement of cAMP and cGMP in the mode of action of molt-inhibiting hormone (MIH) a neuropeptide which inhibits steroidogenesis in a crab.

In crustaceans, production of molting hormones (or ecdysteroids) by the molting glands (Y-organs; YO), is under negative control exerted by a neuropeptide, the molt-inhibiting hormone (MIH). MIH of the crab Carcinus maenas inhibits in vitro steroidogenesis of basal (intermolt crab) or activated (premolt crab) YO. MIH inhibits secretion of the two ecdysteroids synthesized by crab YO, ecdysone (E) secreted throughout the molting cycle, and 25-deoxyecdysone (25dE), secreted during the premolt period. At a MIH concentration of 10(-8) M, E is reduced about 50% and 25dE 94%. Regardless of the molting stage, this inhibition of steroidogenesis is reversible, dose dependent and measurable after 5 min. On intermolt YO, MIH induced cGMP increase and 8BrcGMP mimics the effect of MIH: at this stage cGMP seems to be involved with MIH inhibition of steroidogenesis. On premolt YO MIH induced a transient increase of cAMP (2-fold) and a long-lasting enhancement of cGMP (60-fold). On active YO, we demonstrated that a low concentration (10(-5) M) of dbcAMP, 8BrcAMP, 8BrcGMP, or agents increasing intracellular cAMP, mimic MIH effects and inhibit steroidogenesis. From these observations it is concluded that both cyclic nucleotides are involved in the mode of action of MIH on activated YO. At this premolt period, MIH/cAMP may act cooperatively with MIH/cGMP in the inhibitory control of steroidogenesis by crab YO.

High titers of ecdysteroids are associated with the secretory process of embryonic envelopes in the european lobster.

The newly laid egg of the lobster Homarus gammarus is surrounded by a vitelline coat. Just after fertilization, a new subjacent envelope (2), originating from the cortical reaction, is deposited beneath the vitelline coat. In the course of embryonic development, five new coatings (envelopes 3 to 7) are secreted successively from the ectodermal embryonic cells. These will remain until hatching, freeing the mysis larva in concentric order without exuviation. The concentration of both the two major ecdysteroids (ponasterone A and 20-hydroxyecdysone) and their respective precursors (25-deoxyecdysone and ecdysone) were determined as a function of the secretory phase for three embryonic envelopes (2, 3 and 6). We determined that the secretory processes proceed in the presence of high titers of 20-hydroxyecdysone during the onset of envelope secretion and of ponasterone A in the last phase of secretion.

Studies on the biosynthesis of ecdysone by the Y-organs of Carcinus maenas.

High specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was incubated with Y-organs from intermoult and premoult shore crabs. Several metabolites were identified among which ecdysone and 25-deoxyecdysone. The concomitant production of these 2 molecules by Y-organs and their subsequent hydroxylation at C-20 by peripheral tissues, provide an explanation for the presence of both 20-hydroxyecdysone and ponasterone A (25-deoxy-20-hydroxyecdysone) in the circulating haemolymph of crabs.

Ecdysteroid metabolism in a crab: Carcinus maenas L.

Ponasterone A (25-deoxy-20-hydroxyecdysone) and 20-hydroxyecdysone were the major ecdysteroids detected in crab hemolymph, although some ecdysone was also present. The metabolism of ponasterone A was examined in intermolt and premolt crabs either by injecting the radiolabeled hormone or by incubating tissues in its presence. Metabolites were extracted from the surrounding seawater and from tissues and separated by high-performance liquid chromatography. Ponasterone A metabolism proceeds through (1) C-25 and C-26 hydroxylation, followed by formation of inactivation products via oxidation of the terminal alcoholic group to a carboxylic residue, (2) conjugation, (3) "binding" to very polar compounds and (4) side-chain scission. The conversion of ponasterone A into 20-hydroxyecdysone, inokosterone (25-deoxy-20, 26-dihydroxyecdysone), 20, 26-dihydroxyecdysone and ecdysonoic acids, as well as the formation of conjugates and of very polar compounds, occurs in various tissues. These metabolites were excreted by both intermolt and premolt crabs.

Structure of the egg funiculus and deposition of embryonic envelopes in a crab.

The newly laid egg of Carcinus maenas is attached to a maternal ovigerous seta by a funiculus which consists of the two superimposed vitelline envelopes 1a + 1b, highly stretched and concurrently showing important structural alterations. The funiculus is glued to the specialized seta merely owing to the strong adhesiveness of its external face comprising the outermost vitelline envelope 1a, without any added adhesive. The subjacent envelope 2, originated from the cortical reaction, is not involved in such a funiculus elaboration. In the course of the embryonic development, four new coatings are successively secreted from the ectodermal embryonic cells, underneath the (1a + 1b + 2) fertilization envelope or embryonic capsule. They will remain until hatching in this concentric order, thus giving evidence of successive embryonic moulting cycles, with apolysis but without exuviation. In addition, the successive secretory phases, regarding to the embryonic envelope elaborations, happen in presence of high concentrations of the ecdysteroid ponasterone A which might be involved consequently in such secretory processes.

Ecdysteroids and embryonic development in the shore crab, Carcinus maenas.

Eggs at various stages of embryonic development of Carcinus maenas contain high concentrations of the ecdysteroid ponasterone A together with lower titres of 20-hydroxyecdysone and ecdysone. Correlative studies on ecdysteroid titres and developmental characteristics of Carcinus embryos indicate that one function of ponasterone A might be related to the control of deposition of an embryonic envelope.

Ecdysteroids and ovarian development in the shore crab, Carcinus maenas.

Mature ovaries of the shore crab Carcinus maenas contain large concentrations of three major ecdysteroids which we have identified by physicochemical methods as ecdysone, 20-hydroxyecdysone and ponasterone A. The fluctuations of ovarian and blood ecdysteroid concentrations are presented in relation to the various stages of ovarian development.

Fine structure and secretion of the capsule enclosing the embryo in a crab (Carcinus maenas (L.)).

Carcinus maenas oocyte is encased, at the end of vitellogenesis phase, by two superposed vitelline envelopes. If the outermost (envelope 1a) is relatively thin, the innermost (envelope 1b) is however five times much thicker. The envelope 1b, secreted mostly by the oocyte, contains proteinaceous material and a substance which strongly binds WGA. The 1b material deposition is completed when the oocyte nuclear apparatus still has the characteristics of a germinal vesicle. After the secretion phase and always at the ovarian level, envelope 1b material undergoes remarkable structural modifications. These last events occur while the oocyte nuclear apparatus is in the first meiotic metaphase stage. Later, after egg-laying, a new material appears below the two superimposed vitelline envelopes 1a and 1b. This new innermost material (envelope 2), observed as a very thick and homogeneous secretion, is secreted by the newly laid egg probably after fecondation. Envelope 2 material is wholly deposited in few hours and arises from vesicles of an oocytic origin. These three superimposed envelopes make up the embryo capsule of which the main part is constituted by envelope 2. This tripartite capsule will stay, surrounding the developmental stages, superimposed to newly secreted successive embryonic envelopes, up to the end of embryogenesis.

'Endogenous yolk' as the precursor of a possible fertilization envelope in a crab (Carcinus maenas).

After the egg attachment to a maternal ovigerous seta, the Carcinus maenas embryo is enclosed in a tripartite capsule. The innermost layer (envelope 2) which is also the main part of this capsule, is generally detected after egg-laying and is most probably closely related to the fecondation phenomenon. The precursor material of envelope 2, arising from the egg by a massive and very fast exocytosis process, appears as numerous ring-shaped granules. These granules, originated from numerous cortical vesicles perhaps intercommunicating with each others, are observed early in the ooplasm during oogenesis. These so-called ring-shaped granules seem very identical in form with the disc-shaped granules which are classically described as composing the endogenous or intracysternal yolk of many Decapoda crustacean oocytes. In view of our results the role of these granules, in endogenous yolk formation, is re-examined and discussed.

[Metabolism of ecdysone in several organs of Carcinus maenas L. incubated in vitro]. / Métabolisme de l'ecdysone par divers organes de Carcinus maenas L. incubés in vitro

Tissues of the crab Carcinus maenas in premolt were incubated in vitro with tritiated ecdysone. The digestive tract, the ovaries, the testis the hepatopancreas and epidermis fragments convert into ecdysterone. This conversion, which does not occur in other organs including the Y organs, is the only important in vitro metabolic pathway of ecdysone observed in this study.

[The effect of freezing on the electrophoretic separation of hemolymph proteins of several arthropods]. / Effets de la congélation sur la séparation électrphorétique des protéines de l'hémolymphe de quelques arthropodes

Electrophoresis in 6% or gradient polyacrylamide gel were realised with fresh and stored hemolymphs. The experiments state that there are variations in relation to storage duration, in the separation pattern of hemocyanin fractions in Astacus leptodactylus, Palinurus vulgaris and Carcinus mediterraneus. Moreover, important changements in the supplementary factor of crayfish plasma are revealed.