Identification and properties of a receptor for the invertebrate cytokine astakine, involved in hematopoiesis

We have recently isolated an invertebrate cytokine from a freshwater crayfish, which we named astakine 1. Interestingly this protein is expressed exclusively in hemocytes and hematopoietic tissue and is essential for the release of new hemocytes into the open circulatory system of these animals. This astakine has a prokineticin (PK) domain but lacks the N-terminal AVIT amino acids and hence receptor binding may differ from vertebrate PKs. Accordingly, here we report that a receptor for astakine 1 on hematopoietic tissue (Hpt) cells is identical to the β-subunit of F₁ATP synthase. In this study we have used several different methods to clearly demonstrate that ATP-synthase is located on the plasma membrane of a subpopulation of Hpt cells and there may function as a receptor for astakine, whereas mature blood cells (hemocytes) do not have any ATP-synthase on the outside of their plasma membranes. Our results clearly show that ATP synthase β subunits are present on the cell surface of Hpt cells and highlight the need for more detailed studies on intracellular traffic connections between mitochondria and other membrane compartments.     

Recent advances in crayfish hematopoietic stem cell culture: a model for studies of hemocyte differentiation and immunity

Hematopoiesis is the process by which blood cells (hemocytes) mature and subsequently enter the circulation and we have developed a new technique to culture the hematopoietic progenitor cells in vitro. The reason for the successful culture was the isolation of a plasma protein that turned out to be a novel cytokine, astakine 1 (Ast1) containing a domain present in several vertebrates, so-called prokineticins. Now we have detected several astakines from other invertebrate species. Depending on our discovery of the cytokine Ast1 we have an opportunity to study in detail the differentiation of cells in the hematopoietic tissue of a crustacean, a tissue of evolutionary interest for studies of the connection between the vascular system and the nervous system. We have been able to isolate the entire hematopoietic tissue and for the first time detected a link between this tissue and the brain. We have further localized a proliferation center in the tissue and characterized its different parts. We have also used this system to isolate a new hematopoietic factor CHF that is important in the crossroad between apoptosis and hemocyte differentiation. Our technique for culture of crayfish hematopoietic stem cells provides a simple tool for studying the mechanism of hematopoiesis, but also enables detailed studies of immune defense reactions. Further, the culture system has been used for studies of viral defense and the system is suitable for gene silencing which allows functional characterization of different molecules involved in host defense as well as in hemocyte differentiation.     

PKs的生物学功能

PKs是最近发现的多功能分泌蛋白,由PK1和PK2组成.在不同的系统中,它们通过两个高度同源G-蛋白偶联受体发挥各种生物学功能.它们与神经和血管的形成以及免疫应答的调节有关,并且对生殖系统的正常生理和促性腺激素释放激素系统的发育都有很大的影响.     

鳌虾血细胞中一种新的astakine样造血因子基因的克隆及表达

甲壳动物的血细胞参与多种免疫反应,能够激活酚氧化酶原,产生抗菌肽或进行血细胞的封装、吞噬等. 本研究在克氏原螯虾血细胞中发现一种造血激素astakine,是与脊椎动物细胞因子前动力蛋白prokineticin同源的基因,命名为PcAst,该基因的开放阅读框为357 bp,编码118个氨基酸,具有保守的半胱氨酸残基. 半定量PCR结果显示,该基因只在血细胞中表达. 实时荧光定量PCR结果显示,在金黄色葡萄球菌和鳗弧菌刺激后,其mRNA表达量大幅上调. 白斑综合征病毒刺激后,该基因也呈持续上调表达趋势. 为进一步研究其功能,重组表达了PcAst蛋白,并对其蛋白水平表达模式进行了分析. 所有结果表明,该基因可能参与鳌虾血细胞的固有免疫应答反应,在抗细菌和抗病毒中起重要作用.     

Invertebrate vasopressin/oxytocin homologs. Characterization of peptides from Conus geographus and Conus straitus venoms

The vasopressin-oxytocin family of peptides is of very ancient lineage, found in organisms as diverse as hydra and man. Although these peptides have been intensively studied in vertebrates, the presumably more extensive invertebrate series was defined primarily by immunological methods. In this report, we describe the purification and structures of two peptides of the vasopressin-oxytocin family from molluscs ("Conopressins"), which were found in the venom of fish-hunting marine snails of the genus Conus. The biological activity observed when the two snail peptides are injected intracerebrally into mice is very similar to that elicited by the vertebrate neurohypophyseal hormones and presumably reflects their actions upon a common receptor in the brain. The sequences of the purified peptides reveal unique features not found in the vertebrate peptide series, most notably an additional positive charge. These are the first members of the invertebrate series of the vasopressin-oxytocin family to be characterized biochemically. The sequences of these peptides are: from Conus geographus venom, Lys-conopressin-G, Cys-Phe-Ile-Arg-Asn-Cys-Pro-Lys-Gly-NH2; and from Conus striatus venom, Arg-conopressin-S, Cys-Ile-Ile-Arg-Asn-Cys-Pro-Arg-Gly-NH2.     

Characterization of SoxB2 and SoxC genes in amphioxus (Branchiostoma belcheri): Implications for their evolutionary conservation

Most Sox genes directly affect cell fate determination and differentiation. In this study,we isolated two Sox genes:SoxB2 and SoxC from amphioxus (Branchiostoma belcheri),the closest living invertebrate relative of the vertebrates. Alignments of SoxB2 and SoxC protein sequences and their vertebrate homologs show high conservation of their HMG domains. Phylogenic analysis shows that amphioxus SoxB2 and SoxC fall out of the vertebrate branches,suggesting that vertebrate homologs might arise from gene duplicat...     

Evolution of the chordate muscle actin gene

Summary The ascidians Styela plicata, S. clava, and Mogula citrina are urochordates. The larvae of urochordates are considered to morphologically resemble the ancestral vertebrate. We asked whether larval and adult ascidian muscle actin sequences are nonmusclelike as in lower invertebrates, musclelike as in vertebrates, or possess characteristics of both. Nonmuscle and muscle actin cDNA clones from S. plicata were sequenced. Based on 27 diagnostic amino acids, which distinguish vertebrate muscle actin from other actins, we found that the deduced protein sequences of ascidian muscle actins exhibit similarities to both invertebrate and vertebrate muscle actins. A comparison to muscle actins from different vertebrate and invertebrate phylogenetic groups suggested that the urochordate muscle actins represent a transition from a nonmusclelike sequence to a vertebrate musclelike sequence. The ascidian adult muscle actin is more similar to skeletal actin and the larval muscle actin is more similar to cardiac actin, which indicates that the divergence of the skeletal and cardiac isoforms occurred before the emergence of urochordates. The muscle actin gene may be a powerful probe for investigating the chordate lineage. Offprint requests to: C.R. Tomlinson     

Effects of different vertebrate growth factors on primary cultures of hemocytes from the gastropod mollusc,Haliotis tuberculata

Summary— A useful experimental system from primary cultures of hemocytes from Haliotis tuberculata has been established. Six days after initiation of the culture, the viability of hemocytes remained constant as measured by the MTT assay. In addition, hemocytes showed physiological responses as judged by protein and DNA syntheses in response to treatment with vertebrate growth factors. Porcine insulin and human epidermal growth factor (EGF) stimulated [³H]-leucine and [³H]-thymidine incorporation in hemocytes in a dose-dependent manner. No additive effect of insulin and EGF is observed either for [³H]-leucine or for [³H]-thymidine incorporation. The response of primary cultures of abalone hemocytes to vertebrate growth factors confirms their growth potential in vitro and provides a suitable model for further studies on regulation of the control of cellular processes such as cell growth, differentiation and migration in invertebrate cells.     

A degenerate ParaHox gene cluster in a degenerate vertebrate

The ParaHox genes consist of 3 homeobox gene families, Gsx, Xlox, and Cdx, all of which have fundamental roles in development. Xlox (known as IPF1 or PDX1 in vertebrates), for example, is crucial for development of the vertebrate pancreas and is also involved in regulation of insulin expression. The invertebrate amphioxus has a gene cluster containing one gene from each of the gene families, whereas in all vertebrates examined to date there are additional copies resultant from ParaHox gene cluster duplications at the base of the vertebrate lineage. Extant vertebrates basal to bony and cartilaginous fish are central to the question of when and how these multiple genes arose in the vertebrate genome. Here, we report the mapping of a ParaHox gene cluster in 2 species of hagfishes. Unexpectedly, these basal vertebrates have lost a functional Xlox gene from this cluster, unlike every other vertebrate examined to date. Furthermore, our phylogenetic analyses suggest that hagfishes may have diverged from the vertebrate lineage before the duplications, which created the multiple ParaHox clusters in jawed vertebrates.     

The AVIT protein family. Secreted cysteine-rich vertebrate proteins with diverse functions

Homologues of a protein originally isolated from snake venom and frog skin secretions are present in many vertebrate species. They contain 80–90 amino acids, 10 of which are cysteines with identical spacing. Various names have been given to these proteins, such as mamba intestinal protein 1 (MIT1), Bv8 (Bombina variegata molecular mass ~8 kDa), prokineticins and endocrine-gland vascular endothelial growth factor (EG-VEGF). Their amino-terminal sequences are identical, and so we propose that the sequence of their first four residues, AVIT, is used as a name for this family. From a comparison of the sequences, two types of AVIT proteins can be discerned. These proteins seem to be distributed widely in mammalian tissues and are known to bind to G-protein-coupled receptors. Members of this family have been shown to stimulate contraction of the guinea pig ileum, to cause hyperalgesia after injection into rats and to be active as specific growth factors. Moreover, the messenger RNA level of one of these AVIT proteins changes rhythmically in the region of the brain known as the suprachiasmatic nucleus. This shows that members of this new family of small proteins are involved in diverse biological processes.     

Origin of fibronectin type II (FN2) modules: structural analyses of distantly-related members of the kringle family idey the kringle domain of neurotrypsin as a potential link between FN2 domains and kringles

Analysis of complete genome sequences has made it clear that fibronectin type II (FN2) modules are present only in the vertebrate lineage, raising intriguing questions about the origin of this module type. Kringle domains display many similarities to FN2 domains; therefore it was suggested previously that they are highly divergent descendants of the same ancestral protein-fold. Since kringles are present in arthropodes, nematodes, and invertebrate chordates as well as in vertebrates, it is suggested that the FN2 domain arose in the vertebrate lineage through major structural modification of the more ancestral kringle fold. To explore this structural transition, in the present work we compare key structural features of two highly divergent kringle domains (the kringle of Caenorhabditis elegans Ror receptor tyrosine kinase and the kringle of rat neurotrypsin) with those of plasminogen kringles and FN2 domains. Our NMR conformation fingerprinting analysis indicates that characteristic (1)H-NMR markers of kringle or FN2 native folding, such as the dispersion of Trp aromatic connectivities and shifts of the Leu(46)/Thr(16) methyl signals, both decrease in the order kringles > neurotrypsin kringle > FN2 domains. These results suggest that the neurotrypsin kringle may represent an intermediate form between typical kringles and FN2 domains.     

Evolutionary Analysis of the ErbB Receptor and Ligand Families

We have compared all available deduced protein sequences of the ErbB family of receptors and their ligands. Analysis of the aligned sequences of the receptors indicates that there are some differences in the receptors that are specific to invertebrates. In addition, comparison of the vertebrate ErbB receptors suggest that a gene duplication event generated two ancestral receptors, the ErbB3/ErbB4 precursor and the ErbB1/ErbB2 precursor. Subsequent gene duplications of these precursors generated the four receptors present in mammals. Analysis of the sequences for the known ligands of the ErbB receptors suggests that the vertebrate ligands segregate into the ErbB1 ligands and the ErbB3/ErbB4 ligands, paralleling the evolution of the receptors; however, it is difficult to ascertain any correlation between the invertebrate and the vertebrate ligands. Even though ErbB3 is kinase-impaired, there is significant conservation of the kinase domain within the vertebrate lineage (human, rat, and F. rubripes), suggesting some function for this domain other than kinase activity, such as mediating protein–protein interactions that are involved in receptor dimerization and/or activation of the kinase domain of the heterodimerization partner. To date, no ligand for ErbB2 has been identified, and comparison of the extracellular domains of ErbB2 reveals two regions that are not conserved across the mammalian species. These two regions of divergence align with sequences in ErbB1 that have been shown to be proximal to the amino-terminus and to the carboxyl-terminal region, respectively, of bound EGF. Further, one of these regions contains an insertion, relative to the other members of the mammalian ErbB family, which might affect the ligand binding site and provide a structural basis for this receptor's apparent inability to bind ligand independently. Received: 8 September 1999 / Accepted: 17 January 2000     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

虾类血细胞的分类与功能研究进展

血细胞在动物的免疫防御体系中扮演了重要的角色,尤其是对缺少适应性免疫的无脊椎动物。在这些动物中,血细胞既参与细胞免疫的吞噬、包囊、结节等作用,还参与体液免疫中许多免疫因子的生成与储存。对不同动物类群的血细胞的不同亚群进行区分,是深入了解其免疫机制与血细胞功能的基础。尽管国内外学者利用不同的方法,针对虾类血细胞的不同亚群的区分和功能做了很多研究,但是目前还未形成统一的标准。基于此,文中将已有研究结果进行了梳理与总结,提出了虾类血细胞的三亚群分类法,并对不同亚群的形态特点、免疫学功能进行了详细叙述,期望能为了解无脊椎动物血细胞组成、进行血细胞相关的功能与分子机理研究以及改进和研发新的动物细胞分离技术提供帮助。     

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.     

Insulin-related molecules and insulin effects in the sea urchin embryo

Insulin, the polypeptide hormone secreted by the differentiated pancreas, may play a role in vertebrate development at prepancreatic stages. In an invertebrate embryo, the sea urchin Strongylocentrotus purpuratus, we now find that insulin modulates the levels of developmentally regulated mRNAs of different lineages (one ectoderm-specific, one mesoderm-specific, and one found in all cell types). Using indirect immunofluorescence, we have localized a molecule which shares antigenic determinants with mammalian insulin in the unfertilized egg as well as in the gut of pluteus larva sea urchins. In addition, Southern hybridization reveals high similarity between sea urchin DNA sequences and the human insulin receptor gene. Our results suggest the presence of an insulin/insulin receptor-related system in sea urchin development.