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.

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.

The metallothionein system in Drosophila melanogaster is composed of two genes, Mto and Mtn, that code for distinctly different proteins. In order to compare the properties of Mto and Mm, we transformed yeast with several fusion plasmids. The Mto and Mtn cDNAs, when placed under the control of CUP1 or PGK promoters, can confer a copper-resistance phenotype to copper-hypersensitive cells. Both Mto and Mtn proteins can be characterized in extracts from transformed yeast cells.

Summary 1. In Limulidae, all the factors involved in the coagulation processes are located inside the amoebocytes. The cellular coagulogen is a single 20,000‐polypeptide‐chain protein. It is converted into a non‐covalently crosslinked gel by a serine protease enzyme which cleaves a single peptide bond, releasing peptice C. 2. Pro‐clotting enzyme can be activated by two independent pathways: coagulation is induced by either LPS or 1,3‐β‐D‐glucan, both of which result in gel formation. The two pathways comprise a complex enzyme cascade with several limited protein proteolyses. 3. In Decapoda, clotting factors are found in both the cell‐free plasma and haemocyte compartments. Analogous factors are present in Insecta. 4. Plasma coagulogen is a 400,000 molecular weight protein with both lipid and carbohydrate moieties. Its soluble polymers are converted into covalently crosslinked polymers of coagulin by Ca2+‐dependent transglutaminase. In crayfish, it is also found in other tissues such as soft integument and calcified cuticle. Its concentration varies greatly with the species investigated. It seems to possess many diversified functions such as plasma coagulation, protein transport of tanning agents, lipid and sugar transport and protein storage, and resembles fibronectin. 5. A type of cellular coagulogen seems to be present in the haemocytes of Decapoda. It can be converted to a gel by a serine protease pro‐clotting enzyme. This pro‐enzyme can be activated by either LPS or 1,3‐β‐D‐glucans. The mechanism of LPS action is not entirely clear. 1,3‐β‐D‐glucans also activate the prophenoloxidase system and cause phenoloxidase attachment to foreign surfaces of haemocyte lysates. The latter system is restricted to semi‐granular and granular haemocytes, and plays an important part in host‐defence reactions. 6. The evolutin of clotting processes throughout the phylogenetic tree is discussed.

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 H -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 posttranscriptional mechanisms of APOE regulation in vertebrates.

No abstract

No abstract

Effects of anticoagulants on hemolymph coagulation of lobsters are reported. Effects of some inhibitors on plasma clotting change in relation to molting stages of animals. Variations of electrophoretic and immunochemical protein patterns are analyzed. Complete coagulation inhibition shows that the plasma coagulogen exists as a mixture of soluble uncovalently crosslinked polymers. In lobsters the molecular weight of the lightest visible circulating unit is approximately 420,000 daltons. The weight interval between each of the four lighter polymers is about 200,000 daltons. Serine protease inhibitors impair plasma clotting and interact with cellular clumping.