Retinol binding protein in rainbow trout: molecular properties and mRNA expression in tissues.

Retinoids are important regulatory signaling molecules during embryonic development. The molecular properties of rainbow trout (Oncorhynchus mykiss) retinol-binding protein (rtRBP), the specific retinol carrier in vertebrate plasma, were studied to elucidate its role in transporting retinols to developing fish oocytes. A 954-nucleotide rtRBP cDNA was cloned from the liver coding for a 176-amino-acid (aa) mature protein, with an estimated molecular mass of 20,267 Da. The nucleotide sequence suggests a putative 16-aa signal peptide and shows all the aa residues that were previously identified as critical for the retinol binding pocket. Five of the eight amino acid residues that are associated with the interaction of RBP and transthyretin in mammalian and non-mammalian species are conserved. The deduced aa sequence of rtRBP shows 60-66% identity with zebrafish, chicken, mouse, rat, horse, bovine, and human RBPs and 56% identity with Xenopus RBP. Northern blot analysis revealed a approximately 1.1-kb hepatic mRNA transcript. RBP is highly expressed in the liver, but low levels were also detected in the spleen, kidney, ovary, and brain. In the rainbow trout, 17beta-estradiol treatment led to a decrease in the RBP mRNA signal relative to that of the controls. The efficacy of the 17beta-estradiol treatment was verified by an induction of vitellogenin (VTG) mRNA expression in the liver and occurrence of VTG in the plasma.

Apolipoprotein E gene expression correlates with endogenous lipid nutrition and yolk syncytial layer lipoprotein synthesis during fish development.

During embryogenesis of teleost fish, the formation of a yolk syncytial layer (YSL) enables the resorption of the yolk reserves and development up to the larval stage. We have examined the changes of the yolk cell structure in relation to yolk and oil-globule lipid utilization during development of the turbot (Scophthalmus maximus). After encapsulation by the YSL, resorption of the single, large oil globule occurred predominantly after yolk resorption and was slower in fasting larvae. The YSL was in contact with an enlarged perisyncytial space, but no vascular network or red blood cells were present within the walls of the yolk sac. Intrasyncytial channels infiltrated by pigmented lining cells were observed in the YSL surrounding the oil globule. Apolipoprotein E (apoE) has a prominent role in lipid metabolism because of its ability to interact with lipoprotein receptors. We performed molecular cloning of the putative low-density lipoprotein-receptor binding domain of turbot apoE. In situ hybridization analysis revealed a very high level of apoE transcripts in the YSL, while no expression could be detected in the intestine. YSL apoE expression was correlated with the synthesis of very low density lipoprotein (VLDL) particles. An extraordinarily high number of VLDL particles were poured into the perisyncytial space, and intrasyncytial channels enabled the transfer of yolk- and oil globule-derived lipids to the developing embryo or larva. The pattern of apoE mRNA distribution in relation to YSL lipoprotein synthesis indicates that apoE expression is a suitable molecular marker for monitoring endogenous lipid nutrition during the endoexotrophic period of teleost fish development.

Intestinal fatty acid binding protein gene expression reveals the cephalocaudal patterning during zebrafish gut morphogenesis.

Intracellular fatty acid-binding proteins (FABPs) are small and highly conserved cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. We have examined, as a model for studying intestinal epithelial cell differentiation, the cell-specific and spatio-temporal expression of intestinal fatty acid-binding protein (i-fabp) gene during zebrafish larval development. After molecular cloning of zebrafish I-FABP cDNA, whole-mount in situ hybridization analysis revealed that i-fabp is expressed in the intestinal tube around day 3 postfertilization. By day 4, highest level of i-fabp transcript is encountered in the proximal columnar epithelium. From day 5 onwards, i-fabp is strongly expressed in the anterior intestine and its rostral expansion, slightly expressed in the esophagus mucosa and rectum, while no mRNA could be detected in the posterior intestine. Therefore, the regional differentiation of the intestine precedes first feeding and complete yolk resorption. I-fabp expression in the anterior intestine of the fed larvae is correlated with an intracellular storage of lipid droplets in the enterocytes and the massive synthesis of very low-density lipoprotein particles. In conclusion, the cephalocaudal expression pattern of i-fabp demarcates early during zebrafish gut morphogenesis the anterior fat absorbing to posterior cells of the intestine. This gene could be used as a marker for screening for mutations that affect the events of intestinal epithelial differentiation, cephalocaudal patterning, and asymmetric gut looping morphogenesis.

Conserved protein motifs and structural organization of a fish gene homologous to mammalian apolipoprotein E.

Apolipoprotein E (apoE) plays a central role in lipid metabolism from its ability to interact with lipoprotein receptors. Besides its role in cardiovascular diseases, apoE polymorphism contributes to susceptibility to neurodegenerative diseases, such as Alzheimer's disease. The statistical significance of the combined match scores obtained after apoE motif-based protein sequence database searches, the structural features of the deduced protein, and the phylogenetic analysis, support the evidence that a homologue to mammalian apoE can be found in teleost fish. Isolation and characterization of the first nonmammalian APOE revealed that the zebrafish gene spans 2555/2692 bp instead of 3597 bp in human and has the same splice junctions and exon/intron organization as found in mammals, except that there is an additional intron that splits the last exon (exon 4) into two exons (exons 4 and 5). Enlargement of APOE size in the mammalian lineage occurs mainly by Alu repeats insertion. The additional intron found in zebrafish gene was also identified at the same splicing site in trout APOE and is located in the corresponding linker region following the conserved low density lipoprotein receptor binding domain. Primer extension and reverse transcriptase PCR (RT-PCR) assays demonstrated that two transcription start sites are located 26 and 28 bp upstream of the first intron and 22 or 24 bp downstream from a canonical TATA box. Sequence inspection of the 5'-flanking region upstream of the TATA box revealed potential regulatory DNA elements. These results will serve as a basis for comparative studies on transcriptional and post-transcriptional mechanisms of APOE regulation in vertebrates.

Expression of the Xenopus laevis metallothionein gene during ontogeny.

Expression of the Xenopus laevis metallothionein (MT) gene was studied by in situ hybridization throughout development. MT mRNA was detected from the tailbud stage onwards. MT expression was observed in bucco-pharyngeal epithelium, pronephros and liver anlagen, aswell as in lens and periventricular areas of the encephalon. MT transcripts, in both larvae and adults, were detected in diverse regions of the central nervous system and in differentiating tissues implicated in detoxification processes: liver hepatocytes, small intestine epithelia and kidney tubules. These data are discussed in the context of MT functions and support a physiological role for MT in growth processes.

Both apolipoprotein E and A-I genes are present in a nonmammalian vertebrate and are highly expressed during embryonic development.

Apolipoprotein E (apoE) is associated with several classes of plasma lipoproteins and mediates uptake of lipoproteins through its ability to interact with specific cell surface receptors. Besides its role in cardiovascular diseases, accumulating evidence has suggested that apoE could play a role in neurodegenerative diseases, such as Alzheimer disease. In vertebrates, apoA-I is the major protein of high-density lipoprotein. ApoA-I may play an important role in regulating the cholesterol content of peripheral tissues through the reverse cholesterol transport pathway. We have isolated cDNA clones that code for apoE and apoA-I from a zebrafish embryo library. Analysis of the deduced amino acid sequences showed the presence of a region enriched in basic amino acids in zebrafish apoE similar to the lipoprotein receptor-binding region of human apoE. We demonstrated by whole-mount in situ hybridization that apoE and apoA-I genes are highly expressed in the yolk syncytial layer, an extraembryonic structure implicated in embryonic and larval nutrition. ApoE transcripts were also observed in the deep cell layer during blastula stage, in numerous ectodermal derivatives after gastrulation, and after 3 days of development in a limited number of cells both in brain and in the eyes. Our data indicate that apoE can be found in a nonmammalian vertebrate and that the duplication events, from which apoE and apoA-I genes arose, occurred before the divergence of the tetrapod and teleost ancestors. Zebrafish can be used as a simple and useful model for studying the role of apolipoproteins in embryonic and larval nutrition and of apoE in brain morphogenesis and regeneration.

Ets-1 and Ets-2 proto-oncogenes exhibit differential and restricted expression patterns during Xenopus laevis oogenesis and embryogenesis.

Xenopus XI-ets-1 and XI-ets-2 are maternally expressed. From late oogenesis to early embryogenesis their transcripts are localized to the animal pole and the intermediate zone, suggesting a function in the differentiation of animal blastomeres and future mesoderm. Their presence at the level of germ plasm suggests also a role in the differentiation of the germinal lineage. Both zygotic genes are expressed ubiquitously beginning at MBT, and then restricted to a circumblastoporal collar. In neurula and tailbud stages, ets-1 and ets-2 transcripts are detected in neural crest cells and their derivatives. Specific transcription can also be observed for ets-1 in the hemangioblastic precursors, in endothelial cells of the forming heart and blood vessels. Ets-2 is itself specifically expressed in the putative pronephros and in the forming pronephric tubules and extending pronephric duct. Like another member of the ets-gene family (XI-fli), both genes are transcribed in regions of the embryo undergoing important morphogenetic modifications, especially in migrating cells and/or along their migration pathways. We postulate that these genes orchestrate modifications of cellular adhesion. Changes in the expression of cadherins and integrins repertories would be consistent with such a role and could account for the phenotypes we reported earlier for XI-fli overexpression. Such a role would be critical for tumor cell dissemination, in addition to the one already ascribed to ets-1 in the expression of proteases specific for the extracellular matrix.

Expression of metallothionein genes during the post-embryonic development of Drosophila melanogaster.

Expression of the two Drosophila melanogaster metallothionein genes, Mtn and Mto, has been analyzed by in situ hybridization during post-embryonic development. Mtn and Mto transcripts were detected exclusively in the digestive tract of larvae, pupae and adults reared on standard medium. Mtn and Mto expression domains overlap, but each gene is also expressed at unique sites. Mtn mRNA levels are approximately 10 and 20 times higher than those of Mto in larvae and adults, respectively. Copper and cadmium ions strongly induce Mtn and Mto mRNA accumulation in the midgut. Zinc is a weaker inducer, acting only at high concentrations. Mtn gene expression is induced by these three metals in Malpighian tubules, while Mto gene expression in this organ is induced only by zinc. Iron is a poor inducer of metallothionein mRNA accumulation. Functions of MTN and MTO proteins in metal homeostasis and detoxification are considered.

The c-ets-1 proto-oncogenes in Xenopus laevis: expression during oogenesis and embryogenesis.

We previously reported the cloning and sequencing of two cDNAs derived from the Xenopus laevis ets-1 gene (Stiegler et al., 1990). The Xl-ets-1a cDNA encodes a polypeptide highly homologous to known ets-1 proteins. The 3'-UTR contains two AATAAA polyadenylation signals together with three copies of the TTTTTAT sequence thought to confer a maturation-specific polyadenylation and implicated in the deadenylation of dormant mRNAs. Several transcripts with maternal characteristics were detected in oogenesis and early embryogenesis. A marked augmentation of the major transcript in the poly(A)+ fraction was detected at fertilization. Ets-1 transcripts were observed at constant levels during the cleavage stages but decreased abruptly at gastrulation, to reappear from neurulation to late embryogenesis. The possible contribution of 3'-UTR sequence elements to this behavior is discussed.

Plasminogen receptors on rat colon carcinoma cells.

Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.

A receptor for plasminogen and plasmin on human and rat carcinoma cells. Evidence for strong interspecific reactivity.

We have previously demonstrated the existence of a receptor for plasmin and plasminogen on carcinoma cells, either from human or rat origin. We show here that these receptors have a strong interspecific reactivity. This conclusion is based on binding assay of 125I labelled plasminogens and fluorescence experiments using biotinylated human plasminogen. In all these experiments cells from one species could bind plasminogen from the other species and interspecific inhibition was observed as well.

[Proteases and tumor invasion]. / Protéinases et invasion tumorale.

Degradation of basement membranes is a common feature in the vicinity of carcinomas. It is likely explained by the action of various proteinases, some being produced by tumor cells themselves and some originating in the surrounding tissues. The proteinases which are involved in this lytic process are numerous. Among them one has to stress first plasminogen activators and plasmin and on the other side collagenases. Actually these enzymes seem to have a coordinate action, so as to be described as a proteolytic cascade. Another important factor lies in the existence of proteinases inhibitors. If the balance between proteinases and their inhibitors is altered, basement membranes can be degraded, allowing tumor cells to pass through them, as shown by in vitro experiments.

[Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin]. / Etude immunochimique des protéines de divers tissus chez les Crustacés Décapodes: nature, rôle, origine.

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important rôle in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Coagulation in the crayfish, Astacus leptodactylus: attempts to identify a fibrinogen-like factor in the hemolymph.

1. A series of tests were conducted to determine whether or not the hemolymph of the crayfish Astacus leptodactylus contains a plasma coagulation factor. 2. The total protein amount is higher in the plasma than in the serum. 3. Serum and plasma do not exhibit similar electrophoretic banding patterns. Plasma contains one band or a series of supplementary fractions with a high molecular weight. 4. Electrophoregrams of plasmatic clottable extract, obtained by the classical methods employed in crustacean serology, show a main fraction or a series of polymers with the same electrophoretic behavior as the additional fractions seen on the plasma pattern. 5. This solution clots when treated with CaCl2 and a cellular extract. 6. Immunoelectrophoresis demonstrates the presence of a clottable protein precipitate line in plasma, but this protein also gives a very faint similar line in serum.

[Analysis of the hemocyte proteins from Astacus leptodactylus]. / Analyse des protéines hémocytaires d'Astacus leptodactylus

Three soluble proteins exist in the haemocytes of Astacus leptodactylus in an appreciable amount. The first one is "fibrinogen" apparently in its monomer state, which is at the origin of the clottable plasmatic protein. The second is assumed to be a slow hemocyanin and the third to be a molt fraction.

[Immunologic analysis of the variations of hemolymph proteins of Astacus leptodactylus during the premolt]. / Analyse immunologique des variations des protéines de l'hémolymphe d'Astacus leptodactylus pendant la prémue

The variations in the hemolymph proteins of crayfish were studied by quantitative protein determination, elctrophoresis on polyacrylamide gel gradient and tandem crossed immuno-electrophoresis, from stage C 4 to the end of stage D. The total protein concentration varies during the premolt, with two peaks in D 0 and D I'''-D2, but the plasmatic "fibrinogen" rate is about the same during this period. But three fractions exist which show important changes during the molt cycle.

[Radioautographic study of the action of androgens on the synthesis of nucleic acids in the male genital tract of the white mouse (author's transl)]. / Action des androgènes sur la synthèse des acides nucléiques dans le tractus génital mâle de la souris blanche. Analyse autoradiographique

A comparison was made, by autoradiographic studies, of the nucleic acids synthesis in the target organs of the male hormone, in normal mice, castrate and castrated mice with an implantation of a testosterone pellet. The results show that the response of each of these organs, to a physiological supply of hormone or to a supraphysiological supply is quite different, both in intensity and in evolution. The differences are still apparent in castrated animals. The transfer of RNA from the nucleus to the cytoplasm seems to be affected by the castration. The signification of these results is discussed.

[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.