Identification of antibacterial activity of liver-expressed antimicrobial peptide 2 (LEAP2) from primitive vertebrate lamprey.

Liver-expressed antimicrobial peptide 2 (LEAP2) is a member of the antimicrobial peptides family and plays a key role in the innate immune system of organisms. LEAP2 orthologs have been identified from a variety of fish species, however, its function in primitive vertebrates has not been clarified. In this study, we cloned and identified Lc-LEAP2 from the primitive jawless vertebrate lamprey (Lethenteron camtschaticum) which includes a 25 amino acids signal peptide and a mature peptide of 47 amino acids. Although sequence similarity was low compared to other species, the mature Lc-LEAP2 possesses four conserved cysteine residues, forming a core structure with two disulfide bonds between the cysteine residues in the relative 1-3 (Cys 58 and Cys 69) and 2-4 (Cys 64 and Cys 74) positions. Lc-LEAP2 was most abundantly expressed in the muscle, supraneural body and buccal gland of lamprey, and was significantly upregulated during LPS and Poly I:C stimulations. The mature peptide was synthesized and characterized for its antibacterial activity against different bacteria. Lc-LEAP2 possessed inhibition of a wide range of bacteria with a dose-dependence, disrupting the integrity of bacterial cell membranes and binding to bacterial genomic DNA, although its inhibitory function is weak compared to that of higher vertebrates. These data suggest that Lc-LEAP2 plays an important role in the innate immunity of lamprey and is of great value in improving resistance to pathogens. In addition, the antimicrobial mechanism of LEAP2 has been highly conserved since its emergence in primitive vertebrates.

A Comparative Transcriptomic Study and Key Gene Targeting of Lamprey Gonadal Immune Response.

The mammalian testis and ovary possess special immunocompetence, which is central to provide protection against pathogens. However, the innate immune responses to immune challenges in lamprey gonads are poorly understood. In this study, we extracted RNA from testis and ovary tissues of lampreys at 0 hour, 8 hours and 17 days after lipopolysaccharides (LPS) stimulation and performed transcriptome sequencing. While the transcriptome profiles of the two tissues were different for the most part, genes LIP, LECT2, LAL2, GRN, ITLN, and C1q were found to be the most significantly up-regulated genes in both. Quantitative Real-time PCR (qRT-PCR) analysis confirmed that these genes were upregulated after stimulation. Furthermore, immunohistochemical staining showed that these genes in lamprey gonads are expressed in high quantities and have a specific distribution. Taken together, our results suggest that these genes could play an essential role in response of the gonads to LPS induction. This research establishes a basis for investigating the immune mechanism of vertebrate gonads and presents a fresh concept for gaining insight into the evolutionary development of jawless vertebrates.

Molecular characterization and expression analysis of a novel cold-inducible RNA-binding protein (CIRBP) gene in lamprey (Lethenteron reissneri).

Cold-inducible RNA-binding protein (CIRBP) responds to a wide array of cellular stresses such as cold shock, hypoxia, and inflammatory responses. However, functional studies of CIRBP in jawless vertebrates are limited. In this study, a CIRBP homolog from the jawless vertebrate lamprey (Lethenteron reissneri) was cloned and characterized (named Lr-CIRBP). The cDNA fragment of Lr-CIRBP has a 516 bp open reading frame (ORF) that encodes 171 amino acids, comprising a glycine-rich region at the C-terminal, similar to higher vertebrates but slightly shorter, and an RNA recognition motif (RRM) domain at the N-terminus. The predicted Lr-CIRBP sequence had 51.4 ~ 70.6% similarity with CIRBPs from other vertebrates. Further phylogenetic analysis revealed that Lr-CIRBP is located in the outgroup of vertebrates and is the ancestor of vertebrates. Based on real-time quantitative PCR experimental analysis, Lr-CIRBP expression was highest in leukocytes and increased significantly after multi-stimulation, peaking at 12 h. RNA interference showed that Lr-CIRBP knockdown can down-regulate the expression of inflammatory factors in Lethenteron reissneri. In conclusion, our study successfully clarifies the ancestral features and functions of CIRBP, while revealing valuable insight into how the protein is involved in the immune responses of a jawless vertebrate.

RawHummus: an R Shiny app for automated raw data quality control in metabolomics.

MOTIVATION: Robust and reproducible data is essential to ensure high-quality analytical results and is particularly important for large-scale metabolomics studies where detector sensitivity drifts, retention time and mass accuracy shifts frequently occur. Therefore, raw data need to be inspected before data processing to detect measurement bias and verify system consistency. RESULTS: Here, we present RawHummus, an R Shiny app for an automated raw data quality control (QC) in metabolomics studies. It produces a comprehensive QC report, which contains interactive plots and tables, summary statistics and detailed explanations. The versatility and limitations of RawHummus are tested with 13 metabolomics/lipidomics datasets and 1 proteomics dataset obtained from 5 different liquid chromatography mass spectrometry platforms. AVAILABILITY AND IMPLEMENTATION: RawHummus is released on CRAN repository (https://cran.r-project.org/web/packages/RawHummus), with source code being available on GitHub (https://github.com/YonghuiDong/RawHummus). The web application can be executed locally from the R console using the command 'runGui()'. Alternatively, it can be freely accessed at https://bcdd.shinyapps.io/RawHummus/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Identification and characterization of tryptophan-kynurenine pathway-related genes involving lamprey (Lampetra japonica) innate immunity.

The tryptophan-kynurenine (TRP-KYN) pathway is involved in several biological functions, including immunosuppression, inflammatory response, and tumor suppression. Six TRP-KYN pathway-related genes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 2 (IDO2), aminoadipate aminotransferase (AADAT), glutamate oxaloacetate transaminase 2 (GOT2), kynurenine monooxygenase (KMO), and kynureninase (KYNU) have been identified and cloned from the jawless vertebrate lamprey (Lampetra japonica) to gain insights into their evolution and characterization. Expression distribution showed that the key gene Lj-TDO was highly expressed in the oral gland. Real-time quantitative PCR showed that TRP-KYN pathway-related genes were significantly overexpressed after multi-stimulation. RNA interference showed that Lj-IDO2 knockdown regulated the expression of inflammatory factors. In conclusion, our study successfully clarified the ancestral features and functions of the TRP-KYN pathway, while providing valuable insights into the involvement of this pathway in the immune responses of a jawless vertebrate.

Tryptophan metabolism can modulate immunologic tolerance in primitive vertebrate lamprey via IDO-kynurenine-AHR pathway.

Tryptophan is mainly degraded through kynurenine pathway (KP) in vertebrates which is closely related to the nerve and depression, while the studies on immunity is still limited. This study aims to explore the functions of tryptophan in the innate immunity of primitive vertebrate lamprey. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay showed that tryptophan had no obvious effect on cell viability. Tryptophan was transported into leukocytes and degraded via the KP after tryptophan supplement. Tryptophan treatment (T1x and T2x) failed to alter the total antioxidant capacity regardless of stimulation and exposure time. Real-time quantitative PCR and western blotting results revealed that tryptophan was not only able to reduce the expression of pro-inflammatory factors Lj-TNF-α, Lj-IL1ß and Lj-NF-κB, but also to upregulate the expression of anti-inflammatory factor Lj-TGF-ß independent of stimulation and time. In addition, tryptophan can exert immune tolerance function by inhibiting TLR-MyD88 and promoting (Indoleamine 2, 3-Dioxygenase) IDO-kynurenine-AHR (aryl hydrocarbon receptor) pathways. This study provides a new understanding for tryptophan-kynurenine metabolism and mechanism of immune tolerance function in primitive vertebrate lamprey.

Peptidomics Analysis Reveals the Buccal Gland of Jawless Vertebrate Lamprey as a Source of Multiple Bioactive Peptides.

Various proteins with antibacterial, anticoagulant, and anti-inflammatory properties have been identified in the buccal glands of jawless blood-sucking vertebrate lampreys. However, studies on endogenous peptides in the buccal gland of lampreys are limited. In this study, 4528 endogenous peptides were identified from 1224 precursor proteins using peptidomics and screened for bioactivity in the buccal glands of the lamprey, Lethenteron camtschaticum. We synthesized four candidate bioactive peptides (VSLNLPYSVVRGEQFVVQA, DIPVPEVPILE, VVQLPPVVLGTFG, and VPPPPLVLPPASVK), calculated their secondary structures, and validated their bioactivity. The results showed that the peptide VSLNLPYSVVRGEQFVVQA possessed anti-inflammatory activity, which significantly increased the expression of anti-inflammatory factors and decreased the expression of inflammatory factors in THP-1 cells. The peptide VVQLPPVVLGTFG showed antibacterial activity against some gram-positive bacteria. The peptide VSLNLPYSVVRGEQFVQA possessed good ACE inhibitory activity at low concentrations, but no dose-related correlation was observed. Our study revealed that the buccal glands of the jawless vertebrate lamprey are a source of multiple bioactive peptides, which will provide new insights into the blood-sucking mechanism of lamprey.

A novel serum spherical lectin from lamprey reveals a more efficient mechanism of immune initiation and regulation in jawless vertebrates.

The innate immune system is the body's first line of defense against pathogens and involves antibody and complement system-mediated antigen removal. Immune-response-related complement molecules have been identified in lamprey, and the occurrence of innate immune response via the mannose-binding lectin-associated serine proteases of the lectin cascade has been reported. We have previously shown that lamprey (Lampetra japonica) serum can efficiently and specifically eliminate foreign pathogens. Therefore, we aimed to understand the immune mechanism of lamprey serum in this study. We identified and purified a novel spherical lectin (LSSL) from lamprey serum. LSSL had two structural calcium ions coordinated with conserved amino acids, as determined through cryogenic electron microscopy. LSSL showed high binding capacity with microbial and mammalian glycans and demonstrated agglutination activity against bacteria. Phylogenetic analysis revealed that LSSL was transferred from phage transposons to the lamprey genome via horizontal gene transfer. Furthermore, LSSL was associated with mannose-binding lectin-associated serine protease 1 and promoted the deposition of the C3 fragment on the surface of target cells upon binding. These results led us to conclude that LSSL initiates and regulates agglutination, resulting in exogenous pathogen and tumor cell eradication. Our observations will give a greater understanding of the origin and evolution of the complement system in higher vertebrates and lead to the identification of novel immune molecules and pathways for defense against pathogens and tumor cells.

Genomic analysis and functional characterization of immune genes from the RIG-I- and MAVS-mediated antiviral signaling pathway in lamprey.

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are well-known viral RNA sensors in the cytoplasm. RIG-I-mediated antiviral signals are activated by interacting with the adapter protein mitochondrial antiviral signaling (MAVS), which triggers interferon (IFN) responses via a signaling cascade. Although the complete RIG-I receptor signaling pathway has been traced back to teleosts, definitive evidence of its presence in lampreys is lacking. Here, we identified 13 pivotal molecules in the RIG-I signaling pathway in lamprey, and demonstrated that the original RIG-I/MAVS signaling pathway was activated and mediated the expression of unique immunity factors such as RRP4, to inhibit viral proliferation after viral infection in vivo and in vitro. This study confirmed the conservation of the RIG-I pathway, and the uniqueness of the RRP4 effector molecule in lamprey, and further clarified the evolutionary process of the RIG-I antiviral signaling pathway, providing evidence on the origins of innate antiviral immunity in vertebrates.

Molecular Characterization of a B Cell Adaptor for Phosphoinositide 3-Kinase Homolog in Lamprey (Lampetra japonica) and Its Function in the Immune Response.

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys.

VLRs expression were significantly affected by complement C3 knockdown morphants in Lampetra morii.

The complement component 3 of the lamprey, a jawless vertebrate, functions as an opsonin during the phagocytosis of rabbit red cells. Furthermore, lamprey C3 may be activated and cleaved into C3b, which is attached to the surface of target cells in the cytolytic process. However, the mechanism mediating the biological function of C3 in the lamprey is unknown. To our knowledge, this study is the first to show that variable lymphocyte receptors (VLRs) expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. We identified the C3 gene in the lamprey genome based on its orthologs, conserved synteny, functional domains, phylogenetic tree, and conserved motifs. Additionally, we determined the optimal infection concentration of Aeromonas hydrophila to perform immune stimulation experiments in the lamprey larvae. The quantitative real-time polymerase chain reaction and immunofluorescence analyses revealed that the expression of Lampetra morii C3 (lmC3) was significantly upregulated in the larvae infected with 107 CFU/mL of A. hydrophila. The lmC3 morphants (lmC3 MO) of lamprey larvae were generated by morpholino-mediated knockdown. The lmC3 MO larvae were highly susceptible to A. hydrophila infection, which indicated that lmC3 is critical in lamprey immune response. The expression of a selected panel of orthologous genes was comparatively analyzed in the infected wild type, infected lmC3 MO, infected control MO, uninfected wild type and uninfected lmC3 MO one-month-old ammocoete larvae. The knockdown of lmC3 strongly affected the expression of VLRA+/VLRB+/VLRC+-associated genes, which was also confirmed by immunohistochemical analysis. Thus, VLR expression were significantly affected by complement C3 knockdown morphants in Lampetra morii.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development.

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells.

BACKGROUND: In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. METHODS: Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. RESULTS: We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe209-Gly232 region is predicted to insert into the lipid bilayer to form a transmembrane ß-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. CONCLUSIONS: LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans.

The role of serpin protein on the natural immune defense against pathogen infection in Lampetra japonica.

Serine protease inhibitors (serpins) are a large protein family that is involved in various physiological processes and is known to regulate innate immunity pathways. However, research for the functional study of serpins in lamprey is limited. In the present study, a serpin gene was cloned and characterized from Lampetra japonica at molecular, protein and cellular levels, named L-serpin which belongs to family F serine protease inhibitors (serpin family). The L-serpin includes a serpin domain in the N-terminus. The mRNA transcript of L-serpin was extensively expressed in kidney, supraneural body, intestine, liver, heart, gill and the highest expression in leukocytes. The mRNA expression level of L-serpin increased significantly after Vibrio anguillarum, Staphylocccus aureus and Poly I:C stimulation and dramatically peak at 8 h. It is demonstrated that the L-serpin protected cells from lethal Gram-negative endotoxemia through associating with inhibition of lipopolysaccharide (LPS)-triggered cell death and inflammatory factors expression. Surface plasmon resonance (SPR) and the microbe binding assay were used to determine that L-serpin interacts directly with LPS (KD = 6.14 × 10-7 M). Furthermore, we confirmed L-serpin is a major inhibitor of complement activation by inactivating lamprey-C1q protein (KD = 2.06 × 10-6 M). Taken together, these findings suggest that L-serpin is a endogenous anti-inflammatory factor to defend against Gram-negative bacterial challenge and involved in lamprey innate immunity.

The Anti-Proliferation, Cycle Arrest and Apoptotic Inducing Activity of Peperomin E on Prostate Cancer PC-3 Cell Line.

Peperomin E is a natural secolignan existing distributed in the plants of the genus Peperomia. Previous investigations demonstrated that peperomin E showed potential antitumor activity in some cancer lines, but it is unclear whether peperomin E has an effect on prostate cancer cell lines. The aim of the present study is to investigate its effects on proliferation inhibition, apoptosis-inducing and cell-cycle arrest activity using a prostate cancer PC-3 cell line. The proliferation inhibition was evaluated by MTT assay, apoptosis was detected by Annexin V/propidium iodide (PI) staining and Hoechst 33258 staining, cell cycle distributions were measured by flow cytometry, and western blot analysis was used to determine specific cellular apoptotic protein expressions of Bcl-2, Bax, caspase-3 and cleaved-caspase-3. According to the results of this study, peperomin E exhibited significant anti-proliferation activity on PC-3 cell lines in vitro in a dose-dependent manner. Peperomin E treatments lead to marked morphological changes. Apoptotic cell count and cell-cycle distribution at G2/M phase significantly increased with increasing concentrations of peperomin E. The down-regulated expression level of Bcl-2 and up-regulated expression level of Bax and cleaved-caspase-3 compared with the controls were also observed after peperomin E treatment. These data suggest that peperomin E exhibited proliferation inhabitation, apoptosis-inducing and cell-cycle arrest activity on PC-3 cell lines. The anti-proliferation effect of peperomin E on PC-3 cells should result partly from its cell-cycle arrest and apoptosis-inducing activity, whereas the increasing of the Bax/Bcl-2 ratio and activation of caspases-3 play an important role in the development of apoptosis.

Proteomic analysis of buccal gland secretion from fasting and feeding lampreys (Lampetra morii).

BACKGROUND: Previous studies have shown that lamprey buccal glands contain some regulators related to anticoagulation, nociception, and immune responses due to the blood sucking habit. Regrettably, the protein expression profile in the buccal glands of feeding lampreys has never been reported yet. The present study was performed in order to further identify more proteins which are closely associated with lamprey feeding process. METHODS: 2D-PAGE, NanoLC-MS/MS with higher resolution, Ensembl lamprey and NCBI protein databases, as well as western blot was used to compare the proteomics of buccal gland secretion from China northeast lampreys (Lampetra morii) which had been fed for 0, 10, and 60 min, respectively. RESULTS: In the present study, the number of identified protein species in the buccal glands of feeding groups (60 min) was increased significantly, nearly ten times of that in the fasting group. During the feeding stage, novel proteins emerged in the buccal gland secretion of lampreys. According to gene ontology (GO) analysis and function predictions, these proteins were summarized and discussed based on their potential roles during feeding process. Furthermore, some of the identified proteins were confirmed to express during the feeding time of lampreys. CONCLUSION: When lampreys attack host fishes to suck blood and flesh, their buccal glands could secrete enough proteins to suppress blood coagulation, nociception, oxidative stress, immune response, as well as other adverse effects encountered during their parasitic lives. The present study would provide clues to clarify the feeding mechanism of the bloodsucking lampreys.

[Voltage-gated sodium channels regulate the biological functions of macrophages].

Voltage-gated sodium channels (VGSCs) play very important roles in the generation and conduction of action potential in the excitable cells. Recent studies have showed that VGSCs are also expressed in the macrophages and regulate a variety of biological functions, including phagocytosis, endosomal acidification, podosome formation, polarization, and antiviral responses, etc. This paper will review the roles of VGSCs in regulating the biological functions of macrophages and the underlying mechanisms, which would provide clues for the studies of the functions of VGSCs in the other immune cells.

Correction to: A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells.

CORRECTION: Unfortunately, following publication of this article [1], it was noticed that the key in Figure 5c incorrectly showed '0 h', '5 h' and '10 h'. The corrected version, showing '0 h', '12 h' and '24 h', can be seen below and the original article has been updated to reflect this.

A novel protein derived from lamprey supraneural body tissue with efficient cytocidal actions against tumor cells.

BACKGROUND: In previous research, we found that cell secretion from the adult lamprey supraneural body tissues possesses cytocidal activity against tumor cells, but the protein with cytocidal activity was unidentified. METHODS: A novel lamprey immune protein (LIP) as defense molecule was first purified and identified in jawless vertebrates (cyclostomes) using hydroxyapatite column and Q Sepharose Fast Flow column. After LIP stimulation, morphological changes of tumor cells were analysed and measured whether in vivo or in vitro. RESULTS: LIP induces remarkable morphological changes in tumor cells, including cell blebbing, cytoskeletal alterations, mitochondrial fragmentation and endoplasmic reticulum vacuolation, and most of the cytoplasmic and organelle proteins are released following treatment with LIP. LIP evokes an elevation of intracellular calcium and inflammatory molecule levels. Our analysis of the cytotoxic mechanism suggests that LIP can upregulate the expression of caspase 1, RIPK1, RIP3 to trigger pyroptosis and necroptosis. To examine the effect of LIP in vivo, tumor xenograft experiments were performed, and the results indicated that LIP inhibits tumor growth without damage to mice. In addition, the cytotoxic action of LIP depended on the phosphatidylserine (PS) content of the cell membrane. CONCLUSIONS: These observations suggest that LIP plays a crucial role in tumor cell survival and growth. The findings will also help to elucidate the mechanisms of host defense in lamprey.

A Novel Vav3 Homolog Identified in Lamprey, Lampetra japonica, with Roles in Lipopolysaccharide-Mediated Immune Response.

Vav guanine nucleotide exchange factor 3 (Vav3), a Rho family GTPase, regulates multiple cell signaling pathways including those of T- and B-cell receptors in vertebrates through mediating the activities of the Rho family members. Whether the lamprey possesses Vav3 homolog and what role it plays in immune response remain unknown. Gene cloning, recombinant expression, antibody production and expression pattern analyses were performed to characterize the lamprey Vav3 in the current study. The lamprey Vav3 is closer to jawed vertebrates' Vav3 molecules (about 53% identities in general) than to Vav2 molecules of jawless and jawed vertebrates (about 51% identities in general) in sequence similarity. Conserved motif analysis showed that the most distinguished parts between Vav3 and Vav2 proteins are their two Src-homology 3 domains. The relative expression levels of lamprey vav3 mRNA and protein were significantly up-regulated in lamprey lymphocytes and supraneural myeloid bodies after mixed-antigens stimulation, respectively. In addition, lamprey Vav3 were up-regulated drastically in lymphocytes and supraneural myeloid bodies after lipopolysaccharide (LPS) rather than phytohemagglutinin (PHA) stimulation. Lamprey Vav3 distributed in the cytoplasm of variable lymphocyte receptor B positive (VLRB⁺) lymphocytes, and the number of plasmacytes (VLRB and lamprey Vav3 double positive) in blood lymphocytes also increased after LPS stimulation. Our results proved that lamprey Vav3 was involved in the LPS-mediated immune reaction of lamprey and provided a clue for the further study of the precise role lamprey Vav3 played in the signaling pathway of lamprey VLRB⁺ lymphocytes.