Amphioxus Branchiostoma Belcheri Tsingtauense Research Articles

Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate.

Origin of the phagocytic respiratory burst and its role in gut epithelial phagocytosis in a basal chordate

The vertebrate phagocytic respiratory burst (PRB) is a highly specific and efficient mechanism for reactive oxygen species (ROS) production. This mechanism is mediated by NADPH oxidase 2 (NOX2) and used by vertebrate phagocytic leukocytes to destroy internalized microbes. Here we demonstrate the presence of the PRB in a basal chordate, the amphioxus Branchiostoma belcheri tsingtauense (bbt). We show that using the antioxidant NAC to scavenge the production of ROS significantly decreased the survival rates of infected amphioxus, indicating that ROS are indispensable for efficient antibacterial responses. Amphioxus NOX enzymes and cytosolic factors were found to colocalize in the epithelial cells of the gill, intestine, and hepatic cecum and could be upregulated after exposure to microbial pathogens. The ROS production in epithelial cell lysates could be reconstructed by supplementing recombinant cytosolic factors, including bbt-p47phox, bbt-p67phox, bbt-p47phox, and bbt-Rac; the restored ROS production could be inhibited by anti-bbt-NOX2 and anti-bbt-p67phox antibodies. We also reveal that the gut epithelial lining cells of the amphioxus are competent at bacterial phagocytosis, and there is evidence that the PRB machinery could participate in the initiation of this phagocytic process. In conclusion, we report the presence of the classical PRB machinery in nonvertebrates and provide the first evidence for the possible role of PRB in epithelial cell immunity and phagocytosis.

Identification and primary immune characteristics of an amphioxus akirin homolog

Akirin is a recently described nuclear protein that is thought to be required for the NF-κB signaling pathway in insects and vertebrates. Here, functional investigations of akirin are described in the basal chordate amphioxus Branchiostoma belcheri tsingtauense in an attempt to link this gene between insect and vertebrate lineages. Phylogenetic analysis indicated that amphioxus akirin represented a true ortholog of the two characterized vertebrate akirin paralogs. Amphioxus akirin, coding 219 amino acids with two nuclear localization signal (NLS) sequences and one 14-3-3 binding motif, was widely expressed in various tissues and up-regulated in response to Escherichia coli (Gram-negative bacterium) and Staphylococcus aureus (Gram-positive bacterium) challenges. Furthermore, amphioxus akirin was strictly localized to the nucleus of HEK293T cells in a confocal analysis. Our work identified and characterized for the first time an amphioxus akirin homolog and will promote a better understanding of the evolution and transcriptional network of the akirin gene family.

The conservation and uniqueness of the caspase family in the basal chordate, amphioxus

BackgroundThe caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling.ResultsIn order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as bbtCaspase-8, bbtCaspase-1/2 and bbtCaspase3-like, from the amphioxus Branchiostoma belcheri tsingtauense. We found that bbtCaspase-8 with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while bbtCaspase3-like may mediate a nuclear apoptotic pathway in amphioxus. Also, bbtCaspase-1/2 can co-localize with bbtFADD2 in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that bbtCaspase-1/2 may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis.ConclusionsOur study not only demonstrates the conservation of bbtCaspase-8 in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.

Initiation of primary cell culture from amphioxus Branchiostoma belcheri tsingtauense

Amphioxus, a cephalochordate, is an important model fish for studies in evolution and comparative biology. A successful cell culture from amphioxus tissues in vitro would help understanding some basic issues. To determine the optimal culture conditions for proliferation of amphioxus cells, primary cultures were initiated from buccal cirri, tail, gill, gut and metapleural fold of amphioxus Branchiostoma belcheri tsingtauense. The media tested were L-15, F-12, M 199, MEM, DMEM, PRMI 1640 and LDF, each was supplemented with 20% fetal bovine serum. The optimal conditions include tail tissue cultured in L-15 or F-12 with supplement of 20% FBS and 1.5% NaCl at about 25°C.

Gut-specific expression of cathepsin L and B in amphioxus Branchiostoma belcheri tsingtauense larvae

Cathepsin L and B are involved in yolk processing during oocyte maturation and embryogenesis in most invertebrates and vertebrates. Here we examine the developmental expression of cephalochordate amphioxus cathepsin L (AmphiCL) and cathepsin B (AmphiCB) in the adult and developing embryos and larvae. In situ hybridization of adult amphioxus demonstrated that both AmphiCL and AmphiCB transcripts were strongly expressed in the hepatic caecum and hind-gut besides expression at various levels in other tissues examined. Whole mount in situ hybridization and RT-PCR techniques revealed that AmphiCL and AmphiCB transcripts appeared in the primitive gut in 2-day and 1-day larvae, respectively. However, no signals were detected in the early embryos or larvae. Parallel experiments were performed measuring AmphiCL and AmphiCB activities in the mature oocytes and developing embryos and larvae. Albeit both enzymes were present in the mature oocytes, their roles in yolk protein processing remain to be clarified. In accordance with gene expression patterns, AmphiCL and AmphiCB activities were only detectable in the 2-day larvae, rather than in younger stages, suggesting that both AmphiCL and AmphiCB are implicated in the development of gut, and inferring no roles for yolk processing during embryogenesis in amphioxus Branchiostoma belcheri tsingtauense.

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus ( Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5′-UTR and a 638 bp 3′-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30’s. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.

Characterization, Evolution and Expression of the Calmodulin1 Genes from the Amphioxus <italic>Branchiostoma belcheri tsingtauense</italic>

Two full-length cDNAs, named CaM1a and CaM1b, encoding the highly conserved calmodulin1 (CaM1) proteins, were isolated from the cDNA library of amphioxus Branchiostoma belcheri tsingtauense. There are only two nucleotide differences between them, producing one amino acid difference between CaM1a and CaM1b. Comparison of the amino acid sequence of CaM1 reveals that the B. belcheri tsingtauense CaM1a is identical with CaM1 proteins of B. floridae and B. lanceolatum, Drosophila melanogaster CaM, ascidian Halocynthia roretzi CaMA and mollusk Aplysia californica CaM, and CaM1b differs only at one position (138, Asn to Asp). The phylogenetic analysis indicates that the CaM1 in all three amphioxus species appears to encode the conventional CaM and CaM2 might be derived from gene duplication of CaM1. Southern blot suggests that there are two copies of CaM1 in the genome of B. belcheri tsingtauense. Northern blot and in situ hybridization analysis shows the presence of two CaM1 mRNA transcripts with various expression levels in different adult tissues and embryonic stages in amphioxus B. belcheri tsingtauense. The evolution and diversity of metazoan CaM mRNA transcripts are also discussed.

Antioxidant enzyme activities in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense

Information regarding antioxidant enzymes in amphioxus remains lacking, and this study was carried out to examine the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) in different genders and tissues of amphioxus Branchiostoma belcheri tsingtauense. Results show that (1) CuZn-SOD, CAT and GPX activities in the whole amphioxus B. belcheri tsingtauense were basically at the same levels in male and female amphioxus, whereas both T-SOD and Mn-SOD activities in male amphioxus were significantly higher than that in the female (P<0.05); (2) The testis had significantly higher T-SOD and CuZn-SOD activities than the ovary (P<0.05); (3) CuZn-SOD activity was undetectable in the guts of male and female amphioxus; (4) For both male and female amphioxus, the activities of CAT and GPX in the gonads including testis and ovary were the lowest (P<0.05) among the tissues examined; (5) The gut and gill had the same level GPX activities while the gut had a higher CAT activity; (6) There was no clear difference in CAT and GPX activities in the corresponding tissues between male and female amphioxus. The study on SOD, CAT and GPX activities in different genders and tissues of the protochordate provides data for future comparison of amphioxus antioxidant enzymes with those of invertebrates and vertebrates.

Characterization and copy number of the S27 ribosomal protein gene from amphioxus Branchiostoma belcheri tsingtauense

A cDNA clone encoding ribosomal protein S27 (AmphiS27) was identified in the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. This cDNA consists of 607 bp and contains a 255 bp open reading frame (ORF) corresponding to a deduced protein of 84 amino acids with a calculated molecular mass of 9,488 Da and an isoelectric point (pI) of 9.500. Alignment of the deduced AmphiS27 amino acid sequence with 12 known S27 protein sequences indicates that AmphiS27 shares 94-99% homology with its vertebrate homologue, 84-94% with invertebrate homologues and 69-72% with homologues from other eukaryotes, suggesting that AmphiS27 is more closely related to the vertebrate S27 protein than to its invertebrate counterpart. Southern blot analysis showed a single copy of the S27 gene present in the genome of amphioxus B. belcheri tsingtauense, indicating that amphioxus has a genome uncomplicated by extensive gene duplication.

Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver

Antithrombin (AT), which is mainly synthesized in the liver, is an acute-phase plasma protein in mammalian species. Here, we demonstrated that sheep anti-human AT antibody cross-reacted with the humoral fluids in amphioxus Branchiostoma belcheri tsingtauense as well as human serum. The concentration of AT in the humoral fluids in amphioxus decreased slightly at first and then increased after the acute challenge with lipopolysaccharide, while the level of total proteins remained unchanged. These suggest the presence of the same acute-phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AT was localized in the hepatic diverticulum. It is clear that the hepatic diverticulum in amphioxus is homologous to the vertebrate liver with respect to AT synthesis. This lends support to the hypothesis originally suggested by Müller that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution.

Expression of a novel somite-formation-related gene, AmphiSom, during amphioxus development

A novel gene, AmphiSom, was identified in amphioxus Branchiostoma belcheri tsingtauense. Its sequence and developmental expression pattern were determined. AmphiSom transcripts were first detected in the presomitic mesoderm at the late gastrula stage and reached the highest level in the forming and nascent somites in neurulae. However, the expression of AmphiSom was rapidly down-regulated after somites were formed. It was maintained in the most anterior somite and most posterior somite at neurula stages. By 48 h, AmphiSom transcripts were detected only in the developing tail bud but were no longer detected in 72-h larva. Our data demonstrated that the AmphiSom gene is expressed during the development of somites in amphioxus and could play a role in somite formation.

Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number.

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.

Concerted action between Ca 2+ and hyperosmolality initiates sperm motility in amphioxus Branchiostoma belcheri tsingtauense

This study investigated the effects of different environmental conditions on the initiation and maintenance of sperm motility in amphioxus Branchiostoma belcheri tsingtauense. The findings were that: (1) hyperosmolality in the absence of Ca 2+ ions did not initiate amphioxus sperm motility; (2) addition of Ca 2+ into EGTA-containing Ca 2+-free artificial sea water (ASW), in which no sperm were motile, restored sperm motility; (3) Ca 2+ failed to induce sperm motility under conditions of hypoosmolality; (4) K + channel blockers quinine and 4-aminopiridine did not suppress the initiation of sperm motility; and (5) changes in pH did not cause sperm motility in a solution isotonic to seawater without Ca 2+. In conclusion, we inferred that a concerted action between Ca 2+ and hyperosmolality was essential to initiate motility of amphioxus sperm, whereas K + and pH were indispensable to maintain motility.

Characterization and expression profile ofAmphiCD63encoding a novel member of TM4SF proteins from amphioxusBranchiostoma belcheri tsingtauense

The study on CD antigen genes remains lacking in the cephalochordate amphioxus to date. In this report, the cDNA encoding CD63 was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. Primary structural examination showed that the protein encoded by the cDNA contained four potential transmembrane domains characteristic of transmembrane 4 superfamily (TM4SF) proteins and a conserved CCG motif in the putative major extracellular loop. BLAST search revealed that the cDNA is closely associated with other known CD63 antigen genes, and it was thus designated AmphiCD63. Phylogenetic analysis indicated that AmphiCD63 is extremely close to vertebrate CD63, CD151 and CD53, suggesting they may have been evolved from a common ancestral gene. RT-PCR analysis exhibited that AmphiCD63 mRNA was abundant in muscle, ovary, foregut including hepatic caecum and hindgut, while it was present at considerably lower levels in notochord and gill and absent in testis.

Amphioxus allantoicase: Molecular cloning, expression and enzymatic activity

Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.

G-banding patterns of the chromosomes of amphioxus Branchiostoma belcheri tsingtauense.

Chromosome banding study remains lacking in amphioxus heretofore. This paper reports the G-band patterns of the metaphase chromosomes of amphioxus Branchiostoma belcheri tsingtauense, the first description and numbering of the karyotyped bands in a cephalochordate. G-banding results show that four out of the ten second pair chromosomes examined are heteromorphic, while the remaining ones (6/10) are homomorphic. In contrast, the other 17 pair chromosomes are all homomorphic. These broadly support our previous suggestion that the chromosomes No. 2 are a pair of sex-chromosomes. G-banding method stains positively 65.5% of the chromosome surface, and a total of 149 G-bands (77 positive, 65 negative and 7 variable) has been recorded in the 17 autosomes and the dimorphic chromosomes 2A' and 2A.

Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 °C to 35 °C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca 2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl- p′-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.

C-banding pattern and nucleolar organizer regions of amphioxus Branchiostoma belcheri tsingtauense Tchang et Koo, 1936.

Chromosome banding study remains lacking in amphioxus heretofore. This paper reports the C-band pattern of the metaphase chromosomes of amphioxus Branchiostoma belcheri tsingtauense, the first description of banded karyotype in cephalochordate. The C-banding showed that around 50% of the second chromosome pair examined was heteromorphic, while the remaining ones were homomorphic. In contrast, the other 17 pairs were all homomorphic. These broadly support our previous suggestion that the second chromosome pair is a pair of sex-chromosomes. The C-banding stained positively about 54.3% of the chromosome surface. Most of the centromeric and terminal regions in many chromosomes had positive C-bands, and some interstitial C-bands were also observed in some chromosomes. It seems clear that chromosomes of amphioxus are highly heterochromatinized. In addition, silver staining revealed a single pair of nucleolar organizer regions (NORs) located in the telomeric regions of medium-sized chromosomes. It is suggested that the number and location of NORs in amphioxus represent the primitive condition for chordates.

Identification of the ribosomal proteins s15a and L19 from the amphioxusbranchiostoma belcheri tsingtauense

Abstract The complete cDNA and deduced amino acid sequences of the ribosomal protein S15a (AmphiS15a) and L19 (AmphiL19) of the amphioxus Branchiostoma belcheri tsingtauense were identified in the present study. The AmphiS15a cDNA is 540 nucleotides in length and encodes a 130 amino acid protein with a calculated molecular mass of 149 kDa. It shares 70-85% sequence identity to its known counterparts in the eukaryotes including human, rat, sea urchin, fruit fly and yeast. The AmphiL19 cDNA comprises 728 nucleotides and encodes a 197 amino acid protein with a calculated molecular mass of 23.4 kDa. It shows 59-79% sequence identity to the eukaryotic L19 proteins from human, rat, mouse, fruit fly, Caenorhabditis elegans and yeast Both AmphiS15a and AmphiL19 are highly basic and contain putative nucleolar localization signals. As house-keeping genes, they may both be useful to normalize gene expression in characterization of various genes from the amphioxus.