A Flexible Mouse Model of Autoimmune Thyroiditis Induced by Immunization with an Adenovirus Containing Full-Length Thyroglobulin cDNA.

The main challenge in the "post-GWAS" era is to determine the functional meaning of genetic variants and their contribution to disease pathogenesis. Development of suitable mouse models is critical because disease susceptibility is triggered by complex interactions between genetic, epigenetic, and environmental factors that cannot be modeled by in vitro models. Thyroglobulin (TG) is a key gene for autoimmune thyroid disease (AITD) and several single nucleotide polymorphisms (SNPs) in the TG coding region have been associated with AITD. The classical model of experimental autoimmune thyroiditis (EAT), based on immunization of genetically susceptible mouse strains with purified TG protein in adjuvant, does not allow testing the impact of TG sequence variants on the development of autoimmune thyroiditis. Here we describe a protocol for the induction of EAT by immunization of mice susceptible to thyroiditis with an adenovirus vector carrying full-length human TG cDNA (Ad-TG EAT). We also provide support protocols for evaluation of autoimmune thyroiditis including serological assessment of TG antibodies, in vitro splenocyte proliferation assay and cytokines secretion, thyroid histology, and evaluation of thyroid lymphocytic infiltration by immunostaining. This protocol for EAT induction allows manipulation of the TG cDNA to introduce variants associated with AITD, enabling the testing of the functional effects of susceptible variants and their haplotypes on the immunogenicity of TG. Furthermore, the Ad-TG EAT mouse model is a valuable model for studying the interactions of the TG variants with non-genetic factors influencing AITD development (e.g., cytokines, iodine exposure) or with variants of other susceptible genes (e.g., HLA-DRß1). © 2024 Wiley Periodicals LLC. Basic Protocol: Development of a mouse model of autoimmune thyroiditis induced by immunization with adenovirus containing full-length thyroglobulin cDNA Support Protocol 1: Splenocytes isolation Support Protocol 2: T cell stimulation and carboxyfluorescein diacetate succinimidyl ester (CFSE) based cell proliferation assay Support Protocol 3: Cytokine assays: measuring levels of interferon gamma (IFNγ) and interleukins IL-2, IL-4, and IL-10 in splenocyte supernatants Support Protocol 4: Evaluating thyroid histology and infiltration with immune cells: hematoxylin-eosin staining of mice thyroid glands Support Protocol 5: Immunohistochemistry of thyroid tissues: Immunofluorescence protocol of paraffin-embedded thyroid sections Support Protocol 6: Anti-thyroglobulin antibody measurement in mice sera by enzyme-linked immunosorbent assay (ELISA).

Purification, Characterization, cDNA Cloning, and Bioinformatic Analysis of Zinc-Binding Protein from Magallana hongkongensis.

Oysters contain significant amounts of the zinc element, which may also be found in their proteins. In this study, a novel zinc-binding protein was purified from the mantle of the oyster Magallana hongkongensis using two kinds of gel filtration chromatograms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that its molecular weight was approximately 36 kDa. The protein identified by the Q-Exactive mass spectrometer shared the highest sequence identity with carbonic anhydrase derived from Crassostrea gigas concerning amino acid sequence similarity. Based on homologous cloning and RACE PCR, the full-length cDNA of carbonic anhydrase from Magallana hongkongensis (designated as MhCA) was cloned and sequenced. The cDNA of MhCA encodes a 315-amino-acid protein with 89.74% homology to carbonic anhydrase derived from Crassostrea gigas. Molecular docking revealed that the two zinc ions primarily form coordination bonds with histidine residues in the MhCA protein. These results strongly suggest that MhCA is a novel zinc-binding protein in Magallana hongkongensis.

Expression characteristics of the cyp19a1b aromatase gene and its response to 17ß-estradiol treatment in largemouth bass (Micropterus salmoides).

To investigate the regulatory role of the cyp19a1b aromatase gene in the sexual differentiation of largemouth bass (Micropterus salmoides, LMB), we obtained the full-length cDNA sequence of cyp19a1b using rapid amplification of cDNA ends technique. Tissue expression characteristics and feedback with 17-ß-estradiol (E2) were determined using quantitative real-time PCR (qRT-PCR), while gonad development was assessed through histological section observations. The cDNA sequence of LMB cyp19a1b was found to be1950 base pairs (bp) in length, including a 5' untranslated region of 145 bp, a 3' untranslated region of 278 bp, and an open reading frame encoding a protein consisting of 1527 bp that encoded 508 amino acids. The qRT-PCR results indicated that cyp19a1b abundantly expressed in the brain, followed by the gonads, and its expression in the ovaries was significantly higher than that observed in the testes (P < 0.05). After feeding fish with E2 for 30 days, the expression of cyp19a1b in the pseudo-female gonads (XY-F) was significantly higher than that in males (XY-M) (P < 0.05), whereas expression did not differ significantly between XX-F and XY-F fish (P > 0.05). Although the expression of cyp19a1b in XY-F and XX-F fish was not significantly different after 60 days (P>0.05), both exhibited significantly higher levels than that of XY-M fish (P<0.05). Histological sections analysis showed the presence of oogonia in both XY-F and XX-F fish at 30 days, while spermatogonia were observed in XY-M fish. At 60 days, primary oocytes were abundantly observed in both XY-F and XX-F fish, while a few spermatogonia were visible in XY-M fish. At 90 days, the histological sections' results showed that a large number of oocytes were visible in XY-F and XX-F fish. Additionally, the gonads of XY-M fish contained numerous spermatocytes. These results suggest that cyp19a1b plays a pivotal role in the development of ovaries and nervous system development in LMB.

Molecular identification and development of an infectious cDNA clone of Trichosanthes kirilowii-infecting cucurbit mild mosaic virus.

Trichosanthes kirilowii has been mainly grown for use in traditional Chinese medicine. In this study, cucurbit mild mosaic virus (CuMMV) belonging to the genus Fabavirus was identified from T. kirilowii plants. CuMMV possesses a segmented, bipartite linear single-stranded RNA genome composed of RNA1 and RNA2. Sequence analysis showed that each genomic segment shares the highest sequence similarity with those of CuMMV isolated from pumpkin. A full-length infectious cDNA clone of CuMMV was further constructed and was found to induce typical symptoms in T. kirilowii, Cucumis sativus, C. melo, Citrullus lanatus, and Cucurbita pepo. The sap inoculum derived from the infectious cDNA clone of CuMMV could be mechanically transmitted and reproduce similar symptoms in the tested plants. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of CuMMV, which will provide a useful tool in understanding CuMMV-encoded proteins and plant-CuMMV interactions.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Beet mosaic virus expression of a betalain transcription factor allows visual virus tracking in Beta vulgaris.

In the field of plant virology, the usage of reverse genetic systems has been reported for multiple purposes. One is understanding virus-host interaction by labelling viral cDNA clones with fluorescent protein genes to allow visual virus tracking throughout a plant, albeit this visualization depends on technical devices. Here we report the first construction of an infectious cDNA full-length clone of beet mosaic virus (BtMV) that can be efficiently used for Agrobacterium-mediated leaf inoculation with high infection rate in Beta vulgaris, being indistinguishable from the natural virus isolate regarding symptom development and vector transmission. Furthermore, the BtMV clone was tagged with the genes for the monomeric red fluorescent protein or the Beta vulgaris BvMYB1 transcription factor, which activates the betalain biosynthesis pathway. The heterologous expression of BvMYB1 results in activation of betalain biosynthesis genes in planta, allowing visualization of the systemic BtMV spread with the naked eye as red pigmentation emerging throughout beet leaves. In the case of BtMV, the BvMYB1 marker system is stable over multiple mechanical host passages, allows qualitative as well as quantitative virus detection and offers an excellent opportunity to label viruses in plants of the order Caryophyllales, allowing an in-depth investigation of virus-host interactions on the whole plant level.

Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Molecular cloning and functional analysis of HnSaratin from Hirudo nipponia.

In order to finish a bloodmeal successfully, hematophagous organisms often stored a variety of anticoagulant proteins in their salivary glands, such as proteins that inhibit platelet aggregation. When they ingest a bloodmeal, these proteins are injected into the host to prevent the blood from clotting. As one of the origins of leeches used in traditional Chinese medicine, H. nipponia was proved to be clinically effective in treatment of cardiovascular and cerebrovascular diseases. This study cloned the sequence of HnSaratin cDNA derived from salivary glands of H. nipponia. The sequence contains an open reading frame of 387 bp, encoding a protein of 128 amino acids containing a signal peptide of 21 amino acids. After removal of the signal peptide, the molecular mass of mature HnSaratin was 12.37 kDa, with a theoretical isoelectric point (pI) of 3.89. The N-terminal of mature HnSaratin was folded into a globular structure, in which 3 disulfide bonds, a ßßαßßß topology and 2 Glu residues that binds collagenous Lys2 were located, and the C-terminal formed a flexible region. The fusion HnSaratin protein was obtained by a prokaryotic expression system. The protein showed anti-platelet aggregation activity, and was observed to prevent blood clotting in rats. The significant high expression of HnSaratin mRNA in salivary glands was induced by bloodmeal ingestion of H. nipponia. Briefly, our work provides theoretical basis for further development and utilization of H. nipponia.

A novel Dmrt gene of crustacean: Functional analysis of idmrt-2 gene in the male reproductive system from Scylla paramamosain.

The Dmrt (double-sex and mab-3 related transcription factor) gene family is considered to be a highly conserved gene family related to sex determination and sexual differentiation across species. In order to better understand the role of the idmrt-2 gene in gonad development in Scylla paramamosain, the idmrt-2 gene was cloned and analyzed. The cDNA contains a 1659 bp ORF region encoding 552 amino acids. The qRT-PCR results showed that idmrt-2 was significantly more expressed in the testis than in other tissues (p < 0.05). The expression of idmrt-2 was highest in the spermatids stage (T2 stage), followed by the mature sperms stage (T3 stage) and significantly higher than in the spermatocytes stage (T1 stage) (p < 0.05) during testicular development and the expression difference was not significant in different stages of ovarian development. RNAi studies revealed that after idmrt-2 was knocked down, the expression of Dmrt-like and foxl-2 genes in the testis decreased, as well as IAG expression in the androgenic gland. The findings suggest that idmrt-2 may be an IAG regulator and involved in testicular development.

Novel and diverse mycoviruses co-infecting a single strain of the phytopathogenic fungus Alternaria dianthicola.

Alternaria dianthicola is a pathogenic fungus that causes serious leaf or flower blight on some medicinal plants worldwide. In this study, multiple dsRNA bands in the range of 1.2-10 kbp were found in a Alternaria dianthus strain HNSZ-1, and eleven full-length cDNA sequences of these dsRNA were obtained by high-throughput sequencing, RT-PCR detection and conventional Sanger sequencing. Homology search and phylogenetic analyses indicated that the strain HNSZ-1 was infected by at least nine mycoviruses. Among the nine, five viruses were confirmed to represent novel viruses in the families Hypoviridae, Totiviridae, Mymonaviridae and a provisional family Ambiguiviridae. Virus elimination and horizontal transmission indicated that the (-) ssRNA virus, AdNSRV1, might be associated with the slow growth and irregular colony phenotype of the host fungus. As far as we know, this is the first report for virome characterization of A. dianthus, which might provide important insights for screening of mycovirus for biological control and for studying of the interactions between viruses or viruses and their host.

Cloning, Exogenous Expression and Function Analysis of Interferon-γ from Gadus macrocephalus.

Interferon γ (IFN-γ) is now considered to be one of the key molecules in the regulation of innate and adaptive immunity. The function of IFN-γ is best described in humans, but less of IFN-γ in fish species has been described at protein level. In the present study, IFN-γ from Gadus macrocephalus (GmIFN-γ) has been examined in terms of bioinformatics, prokaryotic expression, yeast expression, antiviral activity and immune regulatory function. The cDNA of GmIFN-γ contains an open reading frame of 570 nucleotides, coding 189 amino acids. The mature protein contains a nuclear localization signal motif and an obvious IFN-γ signature sequence at the C-terminal. GmIFN-γ is very similar to that of Atlantic cod, with homology up to 89.89%, but less than 32% to other species. GmIFN-γ can be detected in the gills, spleen, intestine, brain and kidney. Interestingly, during early development, a strong signal of GmIFN-γ was not detected until 40 days post hatching. Prokaryotic expression plasmid pET-32a-GmIFN-γ was constructed, and the expression products in BL21 were confirmed by Mass Spectrometry. Meanwhile, the plasmid pGAPZA-GmIFN-γ with Myc tag was constructed and transmitted into Pichia pastoris yeast GS115, and the products were tested using Western blot. The purified GmIFN-γ from either BL21 or yeast has a strong antivirus (Spring viremia of carp virus) effect. The vector of pcDNA3.1-GmIFN-γ was expressed in EPC cell lines; high transcript levels of MHC class I chain-related protein A (MICA) gene were detected; and the exogenous GmIFN-γ protein could also induce MICA expression, indicating that GmIFN-γ could stimulate immune response. The yeast GS115 with GmIFN-γ protein, which is an inclusion body, was given to zebrafish orally, and the transcript of zebrafish IFN-γ was upregulated significantly; however, genes of the interferon type-I signal pathway were not well stimulated.

Cloning, Expression, and Functional Analysis of the Full-Length cDNA of Acetyl-CoA C-acetyltransferase (AACT) Genes Related to Terpenoid Synthesis in Platycodon grandiflorus.

Platycodon grandiflorus is a well-known and widely distributed traditional herbal medicine and functional food in Asia, with triterpenoids as the main bioactive component in its roots. Acetyl-CoA C-acetyltransferase (AACT) is the initiation enzyme in the mevalonate pathway and plays an important role in the biosynthesis of terpenoids. OBJECTIVE: The objective of this study was to clone and identify the PgAACT function in P. grandiflorus. METHODS: The full-length sequence of PgAACT genes was isolated and cloned from P. grandiflorus by polymerase chain reaction (PCR). The recombinant plasmid was constructed using the pET-32a vector and expressed in E. coli Transetta (DE3) cells. Subcellular localization of AACT was observed in the epidermal cells of N. tabacum. Quantitative reverse transcription-PCR (qRT-PCR) was used to identify the PgAACT gene transcription levels. After MeJA treatment, the changes in AACT gene expression were observed, and UHPLC-Q-Exactive Orbitrap MS/MS was used to detect the changes in P. grandiflorus saponins. RESULTS: In this study, two full-length cDNAs encoding AACT1 (PgAACT1) and AACT2 (PgAACT2) were isolated and cloned from P. grandiflorus. The deduced PgAACT1 and PgAACT2 proteins contain 408 and 416 amino acids, respectively. The recombinant vectors were constructed, and the protein expression was improved by optimizing the reaction conditions. Sodium dodecyl sulphate-polycrylamide gel electrophloresis and western blot analysis showed that the PgAACT genes were successfully expressed, with molecular weights of the recombinant proteins of 61 and 63 kDa, respectively. Subcellular localization showed that the PgAACT genes were localized in the cytoplasm. Tissue specificity analysis of P. grandiflorus from different habitats showed that PgAACT genes were expressed in the roots, stems, and leaves. After MeJA treatment, the expression level of PgAACT genes and the content of total saponins of P. grandiflorus were significantly increased, suggesting that PgAACT genes play an important role in regulating plant defense systems. CONCLUSION: Cloning, expression, and functional analysis of PgAACT1 and PgAACT2 will be helpful in understanding the role of these two genes in terpene biosynthesis.

Expression of ABCA3 transporter gene in Tegillarca granosa and its association with cadmium accumulation.

Exposure to cadmium (Cd), a heavy metal, can cause strong and toxic side effects. Cd can enter the body of organisms in several ways, leading to various pathological reactions in the body. Tegillarca granosa is a kind of bivalve shellfish favored by people in the coastal areas of China. Bivalve shellfish can easily absorb heavy metal pollutants from water bodies while filter feeding. T. granosa is considered a hyper-accumulator of Cd, and the TgABCA3 gene is highly expressed in individuals with a high content of Cd-exposed blood clam. However, it is unclear whether TgABCA3 is involved in Cd ion transport in blood clam and the molecular mechanism for the mechanism of the Cd-induced responses for maintaining cell homeostasis. In this study, the complete cDNA of the TgABCA3 gene was analyzed to provide insights into the roles of TgABCA3 in resistance against Cd in blood clam. The complete sequence of TgABCA3 showed high identity to that of TgABCA3 from other bivalves and contained some classical motifs of ATP-binding cassette transport proteins. TgABCA3 expression in different tissues was measured using real-time quantitative polymerase chain reaction (qRT-PCR) and western blot analysis. The tissue-specific expression showed that TgABCA3 expression was highest in the gill tissue. The TgABCA3 expression in the gill tissue was silenced using the RNA interference technique. After TgABCA3 silencing, the TgABCA3 expression decreased, the Cd content increased, the oxygen consumption and ammonia excretion rates increased, and the ingestion rate decreased. These results showing that the extents of Cd accumulation and resulting toxic effects are related to expression levels and activity of TgABCA3 indicate that TgABCA3 has a protective function against Cd in the clam. This increase in Cd accumulation results in serious damage to the body, leading to the enhancement of its physiological metabolism. Therefore, the findings of the study demonstrated that TgABCA3 can participate in the transport of Cd ions in the blood clam through active transport and play a vital role in Cd detoxification.

Molecular characterization of the evolutionary conserved signaling intermediate in Toll pathways (ECSIT) of soiny mullet (Liza haematocheila).

Mammalian evolutionary conserved signaling intermediate in Toll pathways (ECSIT) is an important intracellular protein that involves in innate immunity, embryogenesis, and assembly or stability of the mitochondrial complex I. In the present study, the ECSIT was characterized in soiny mullet (Liza haematocheila). The full-length cDNA of mullet ECSIT was 1860 bp, encoding 449 amino acids. Mullet ECSIT shared 60.4%∼78.2% sequence identities with its teleost counterparts. Two conserved protein domains, ECSIT domain and C-terminal domain, were found in mullet ECSIT. Realtime qPCR analysis revealed that mullet ECSIT was distributed in all examined tissues with high expressions in spleen, head kidney (HK) and gill. Further analysis showed that mullet ECSIT in spleen was up-regulated from 6 h to 48 h after Streptococcus dysgalactiae infection. In addition, the co-immunoprecipitation (co-IP) assay confirmed that mullet ECSIT could interact with tumor necrosis factor receptor-associated factor 6 (TRAF6). Molecular docking revealed that the polar interaction and hydrophobic interaction play crucial roles in the forming of ECSIT-TRAF6 complex. The resides of mullet ECSIT that involved in the interaction between ECSIT and TRAF6 were Arg107, Glu113, Phe114, Glu124, Lys120 and Lys121, which mainly located in the ECSIT domain. Our results demonstrated that mullet ECSIT involved in the immune defense against bacterial and regulation of TLRs signaling pathway by interaction with TRAF6. To the best of our knowledge, this is the first report on ECSIT of soiny mullet, which deepen the understanding of ECSIT and its functions in the immune response of teleosts.

Characterization and Expression Analysis of Regeneration-Associated Protein (Aj-Orpin) during Intestinal Regeneration in the Sea Cucumber Apostichopus japonicus.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand_7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

Comparative analysis of virus pathogenicity and resistance-breaking between the P- and A-type from the beet necrotic yellow vein virus using infectious cDNA clones.

The A-type of beet necrotic yellow vein virus (BNYVV) is widely distributed in Europe and is one of the major virus types causing rhizomania disease in sugar beet. The closely related P-type is mainly limited to a small region in France (Pithiviers). Both virus types possess four RNAs (RNA1-4), but the P-type harbours an additional fifth RNA species (RNA5). The P-type is associated with stronger disease symptoms and resistance-breaking of Rz1, one of the two resistance genes which are used to control BNYVV infection. These characteristics are presumably due to the presence of RNA5, but experimental evidence for this is lacking. We generated the first infectious cDNA clone of BNYVV P-type to study its pathogenicity in sugar beet in comparison to a previously developed A-type clone. Using this tool, we confirmed the pathogenicity of the P-type clone in the experimental host Nicotiana benthamiana and two Beta species, B. macrocarpa and B. vulgaris. Independent of RNA5, both the A- and the P-type accumulated in lateral roots and reduced the taproot weight of a susceptible sugar beet genotype to a similar extent. In contrast, only the P-type clone was able to accumulate a virus titre in an Rz1-resistant variety whereas the A-type clone failed to infect this variety. The efficiency of the P-type to overcome Rz1 resistance was strongly associated with the presence of RNA5. Only a double resistant variety, harbouring Rz1 and Rz2, prevented an infection with the P-type. Reassortment experiments between the P- and A-type clones demonstrated that both virus types can exchange whole RNA components without losing the ability to replicate and to move systemically in sugar beet. Although our study highlights the close evolutionary relationship between the two virus types, we were able to demonstrate distinct pathogenicity properties that are attributed to the presence of RNA5 in the P-type.

Cloning of Metalloproteinase 17 Genes from Oriental Giant Jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae).

We previously demonstrated that Nemopilema nomurai jellyfish venom metalloproteinases (JVMPs) play a key role in the toxicities induced by N. nomurai venom (NnV), including dermotoxicity, cytotoxicity, and lethality. In this study, we identified two full-length JVMP cDNA and genomic DNA sequences: JVMP17-1 and JVMP17-2. The full-length cDNA of JVMP17-1 and 17-2 contains 1614 and 1578 nucleotides (nt) that encode 536 and 525 amino acids, respectively. Putative peptidoglycan (PG) binding, zinc-dependent metalloproteinase, and hemopexin domains were identified. BLAST analysis of JVMP17-1 showed 42, 41, 37, and 37% identity with Hydra vulgaris, Acropora digitifera, Megachile rotundata, and Apis mellifera venom metalloproteinases, respectively. JVMP17-2 shared 38 and 36% identity with H. vulgaris and A. digitifera, respectively. Alignment results of JVMP17-1 and 17-2 with other metalloproteinases suggest that the PG domain, the tissue inhibitor of metalloproteinase (TIMP)-binding surfaces, active sites, and metal (ion)-binding sites are highly conserved. The present study reports the gene cloning of metalloproteinase enzymes from jellyfish species for the first time. We hope these results can expand our knowledge of metalloproteinase components and their roles in the pathogenesis of jellyfish envenomation.

Tollip suppresses MyD88-mediated NF-κB activation by enhancing MyD88 ubiquitination levels in large yellow croaker (Larimichthys crocea).

Toll-interacting protein (Tollip) plays an important role in the innate immune response by negative regulation of the TLR-IL-1R signaling pathway. MyD88 serves as a universal adaptor in TLR-mediated NF-κB activation. However, the regulation mechanisms of Tollip in piscine MyD88-mediated NF-κB activation is largely unknown. In the present study, the cDNA sequence of LcTollip was identified from the large yellow croaker (Larimichthys crocea). The putative LcTollip protein encoded 275 amino acid residues, containing a N-terminal TBD domain, a central C2 domain, and a C-terminal CUE domain. Quantitative PCR showed that the most predominant constitutive expression of LcTollip was detected in spleen. In addition, LcTollip transcripts enhanced significantly after LPS and poly I:C challenge (P < 0.05). Cellular localization revealed that LcTollip existed in the cytoplasm and nucleus. Furthermore, the overexpression plasmids of wild type LcTollip as well as its six domain truncated mutants of LcTollip were constructed by overlap PCR. Dual luciferase analysis showed that NF-κB activation could not be induced by overexpression of LcTollip or its domain truncated mutants alone. However, the LcMyD88-induced-NF-κB activation was significantly suppressed by overexpression with LcTollip, and the truncated mutants LcTollip-ΔTBD, LcTollip-ΔC2, LcTollip-ΔCUE and LcTollip-ΔTBDΔCUE while not by LcTollip-ΔLR and LcTollip-ΔTBDΔC2. Moreover, co-immunoprecipitation (Co-IP) assay revealed that the interaction between LcTollip and LcMyD88 was through CUE domain. More interesting, IP and immunoblotting examination of HEK293T cells co-transfected with LcMyD88, LcTollip and HA-ubiquitin showed that LcMyD88 induced a dose-dependent de-ubiquitination of LcTollip while LcTollip enhanced a dose-dependent ubiquitination of LcMyD88. However, protein degradation investigation displayed that the proteolysis and ubiquitination of LcMyD88 were not connected. Our findings suggested that the LcTollip might involve in negative regulation TLR pathway by suppressing LcMyD88-mediated immune activation and improving the ubiquitination level of LcMyD88.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.

An extracellular Cu/Zn superoxide dismutase from Neocaridina denticulata sinensis: cDNA cloning, mRNA expression and characterizations of recombinant protein.

Neocaridina denticulata sinensis possesses characters of rapid growth, tenacious vitality, short growth cycle, transparent, and easy feeding. Therefore, it is gradually being developed into an animal model for basic research on decapod crustaceans. Herein, a Cu/Zn superoxide dismutase (Cu/Zn-SOD), named as Nd-ecCu/Zn-SOD, was identified and characterized from N. denticulata sinensis. The full-length cDNA sequence of Nd-ecCu/Zn-SOD is 829 bp containing a 684 bp open reading frame, which encodes a protein of 227 amino acid residues with a typical Sod_Cu domain. The quantitative real-time PCR analysis showed that Nd-ecCu/Zn-SOD mRNA was expressed in all the tested tissues. Under challenge with copper, the mRNA expression of Nd-ecCu/Zn-SOD reached the maximum at 6 h, and decreased until 24 h. After 24 h of exposure, its expression was up-regulated significantly at 36 h. After then its expression sharply decreased with a comeback at 48 h. The result indicated that Nd-ecCu/Zn-SOD might play an important role in the stress response of N. denticulata sinensis. The expression of Nd-ecCu/Zn-SOD in gills challenged with Vibrio parahaemolyticus changed in a time-dependent manner. Nd-ecCu/Zn-SOD was lowly expressed in early developmental stages by RNA-Seq technology, yet it showed that a cyclical rise and fall occurred between middle stages and late stages. In addition, Nd-ecCu/Zn-SOD was recombinantly expressed using E. coli and the recombinant protein was purified as a single band on SDS-PAGE. The recombinant Nd-ecCu/Zn-SOD (rNd-ecCu/Zn-SOD) existed enzymatic activity under a wide range of temperature and pH. The exposure of metal ions was found that Zn2+, Mg2+, Ca2+, Ba2+, and Cu2+ could inhibit the enzymatic activity of rNd-ecCu/Zn-SOD, and Mn2+ increased the enzymatic activity of rNd-ecCu/Zn-SOD. These results indicate that Nd-ecCu/Zn-SOD may play a pivotal role in resistant against oxidative damage and act as a biomarker under stressful environment.