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BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.
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Proteínas de Anfíbios , Antibacterianos , Filogenia , Ranidae , Animais , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/química , Proteínas de Anfíbios/genética , Camundongos , Antibacterianos/farmacologia , Antibacterianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Sequência de Aminoácidos , Pele/metabolismo , Fatores Imunológicos/farmacologia , Fatores Imunológicos/química , Células RAW 264.7 , Alinhamento de SequênciaRESUMO
Acute pancreatitis (AP) is a frequent abdominal inflammatory disease. Despite the high morbidity and mortality, the management of AP remains unsatisfactory. Disulfiram (DSF) is an FDA-proved drug with potential therapeutic effects on inflammatory diseases. In this study, we aim to investigate the effect of DSF on pancreatic acinar cell necrosis, and to explore the underlying mechanisms. Cell necrosis was induced by sodium taurocholate or caerulein, AP mice model was induced by nine hourly injections of caerulein. Network pharmacology, molecular docking, and molecular dynamics simulation were used to explore the potential targets of DSF in protecting against cell necrosis. The results indicated that DSF significantly inhibited acinar cell necrosis as evidenced by a decreased ratio of necrotic cells in the pancreas. Network pharmacology, molecular docking, and molecular dynamics simulation identified RIPK1 as a potent target of DSF in protecting against acinar cell necrosis. qRT-PCR analysis revealed that DSF decreased the mRNA levels of RIPK1 in freshly isolated pancreatic acinar cells and the pancreas of AP mice. Western blot showed that DSF treatment decreased the expressions of RIPK1 and MLKL proteins. Moreover, DSF inhibited NF-κB activation in acini. It also decreased the protein expression of TLR4 and the formation of neutrophils extracellular traps (NETs) induced by damage-associated molecular patterns released by necrotic acinar cells. Collectively, DSF could ameliorate the severity of mouse acute pancreatitis by inhibiting RIPK-dependent acinar cell necrosis and the following formation of NETs.
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Pancreatite , Camundongos , Animais , Pancreatite/tratamento farmacológico , Pancreatite/induzido quimicamente , Células Acinares , Dissulfiram/efeitos adversos , Ceruletídeo/efeitos adversos , Doença Aguda , Simulação de Acoplamento Molecular , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/uso terapêuticoRESUMO
Taiwania cryptomerioides Hayata is an endangered relict plant belonging to Taxodiaceae, and it is also an endemic plant to China. The decay-resistant of Taiwania timber can provide highly quality wood for building and furniture. Plenty of regenerative of leaves of T.â cryptomerioides also has been used as a resource for the discovery of new dimeric diterpenoids. In a search for structurally diverse dimeric diterpenoids and potent bioactive isolates, ten new heterodimeric diterpenoids, taiwaniadducts K-T (1-4, 6, 8-11, and 14), along with five known ones (5, 7, 12, 13, and 15), were isolated from the leaves of T.â cryptomerioides. These new compounds were defined by comprehensive spectroscopic analyses, putative biosynthetic pathways, and the values of optical. Biologically, anti-multidrug resistance (MDR) activities of compounds were evaluated. Compounds 4 and 10 exerted a 9.18-fold potentiation effect on bortezmib (BTZ) susceptibility at a tested concentration (20â µM) better than the positive control verapamil. The research of the leaves of T.â cryptomerioides not only added the new data to the structural diversity and activities of dimeric diterpenoids but also could provide support for the medical and industrial application of the leaves of this endangered relict plant.
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Cupressaceae , Diterpenos , Diterpenos/química , Extratos Vegetais/química , Madeira , Análise Espectral , Cupressaceae/química , Estrutura MolecularRESUMO
BACKGROUND: The tiger frog (Hoplobatrachus rugulosus) is listed as a national Class II protected species in China. In the context of global warming, the sex ratio of amphibians will be affected, and the development of the population will be limited. Therefore, considering the potential for a decrease in the number of amphibians, studying sex evolution and molecular regulation of gonadal development in H. rugulosus, phenomenon that are currently unclear, is of great significance. RESULTS: Here, H. rugulosus was used to explore the mechanisms regulating gonadal development in amphibians. Illumina HiSeq 3000 was used to sequence the gonadal transcriptome of male and female H. rugulosus at two growth stages to identify genes related to gonadal development and analyze expression differences in the gonads. This analysis indicated that cyp17α, hsd3ß, hsd11ß1, cyp19α, and hsd17ß12 perform vital functions in sex development in amphibians. Specifically, the expression of cyp3α, cyp17α, hsd3ß, hsd11ß1, sox2, sox9, sox30, soat, cyp19α, hsd17ß12, and hspα1s was correlated with gonadal development and differentiation in H. rugulosus, as determined using the quantitative reverse transcriptase-polymerase chain reaction. CONCLUSION: Significant differences were found in the gonadal gene expression levels in H. rugulosus of both sexes, and we identified a steroid hormone synthesis pathway in this species and analyzed related gene expression, but the changes during sex differentiation were still unclear. To our knowledge, this report presents the first analysis of the H. rugulosus gonadal transcriptome and lays the foundation for future research.
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Gônadas , Transcriptoma , Animais , Anuros/genética , China , Feminino , Masculino , Diferenciação Sexual/genética , Desenvolvimento SexualRESUMO
Serotonin (5-HT) receptors are proteins involved in various neurological and biological processes, such as aggression, anxiety, appetite, cognition, learning, memory, mood, sleep, and thermoregulation. They are commonly associated with drug abuse and addiction due to their importance as targets for various pharmaceutical and recreational drugs. However, due to a high sequence similarity/identity among 5-HT receptors and the unavailability of the 3D structure of the different 5-HT receptor, no report was available so far regarding the systematical comparison of the key and selective residues involved in the binding pocket, making it difficult to design subtype-selective serotonergic drugs. In this work, we first built and validated three-dimensional models for all 5-HT receptors based on the existing crystal structures of 5-HT1B, 5-HT2B, and 5-HT2C. Then, we performed molecular docking studies between 5-HT receptors agonists/inhibitors and our 3D models. The results from docking were consistent with the known binding affinities of each model. Sequentially, we compared the binding pose and selective residues among 5-HT receptors. Our results showed that the affinity variation could be potentially attributed to the selective residues located in the binding pockets. Moreover, we performed MD simulations for 12 5-HT receptors complexed with ligands; the results were consistent with our docking results and the reported data. Finally, we carried out off-target prediction and blood-brain barrier (BBB) prediction for Captagon using our established hallucinogen-related chemogenomics knowledgebase and in-house computational tools, with the hope to provide more information regarding the use of Captagon. We showed that 5-HT2C, 5-HT5A, and 5-HT7 were the most promising targets for Captagon before metabolism. Overall, our findings can provide insights into future drug discovery and design of medications with high specificity to the individual 5-HT receptor to decrease the risk of addiction and prevent drug abuse.
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Receptores de Serotonina/metabolismo , Antagonistas da Serotonina/metabolismo , Agonistas do Receptor de Serotonina/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Farmacologia/métodos , Receptores de Serotonina/química , Antagonistas da Serotonina/química , Agonistas do Receptor de Serotonina/químicaRESUMO
Recently, populations of Chinese spiny frogs (Quasipaa spinosa), an important amphibian species in China, have decreased, mainly due to a disease caused by the gram-negative bacteria Proteus mirabilis. To elucidate the immune response of the frogs, this study aimed to identify novel candidate genes functionally associated with P. mirabilis infection-induced "rotting skin" disease. Chinese spiny frogs were infected with P. mirabilis, and the skin transcriptome was sequenced using the MGISEQ-2000 platform. A total of 233,965 unigenes were obtained by sequencing, of which 27.23 % were known genes. Screening of differentially expressed genes (DEGs) indicated 210 unigenes differentially expressed after P. mirabilis infection, of which 132 unigenes were up-regulated, and 78 unigenes were down-regulated. Using Kyoto Encyclopedia of Genes and Genomes enrichment analysis, DEGs were identified as enriched in signal pathways, such as oxidative phosphorylation, apoptosis, and the Janus kinase-signal transducer and activator of transcription pathway. Of the DEGs, there was a significant upregulation of the colony stimulating factor 2 receptor beta common subunit, interleukin 2 receptor subunit gamma, cathelicidin antimicrobial peptide, interleukin-17 receptor E, receptor-interacting serine/threonine-protein kinase 3, and pulmonary surfactant-associated protein D immune genes following P. mirabilis infection. Conversely, scavenger receptor cysteine-rich domain-containing group B protein, tumor protein p53 inducible nuclear protein 2, suppressor of cytokine signaling 2, and metalloreductase STEAP3 were significantly downregulated. In conclusion, the first skin transcriptome database of Chinese spiny frogs was established, and several immune genes were identified to elucidate the pathogenic mechanism of "skin rot" in Chinese spiny frogs and other cultured frogs.
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Proteus mirabilis , Dermatopatias , Animais , Proteus mirabilis/genética , Perfilação da Expressão Gênica , Transcriptoma , Anuros , Ranidae/genéticaRESUMO
Cathelicidins are important antimicrobial peptides in various vertebrate species where they are crucial parts of the innate immune system. The current understanding of amphibian cathelicidins is limited, particularly with regard to their immunomodulatory effects. To address this knowledge gap, we produced the cDNA sequence of the cathelicidin gene from a skin transcriptome of the Chinese spiny frog Quasipaa spinosa. The amino acid sequence of the Quasipaa spinosa cathelicidin (QS-CATH) was predicted to consist of a signal peptide, a cathelin domain, and a mature peptide. Comparative analysis of the QS-CATH amino acid sequence with that of other amphibian cathelicidins revealed high variability in the functional mature peptide among amphibians, whereas the cathelin domain was conserved. The QS-CATH gene was expressed in several tissues, with the highest level of expression in the spleen. Upregulation of QS-CATH after Aeromonas hydrophila infection occurred in the kidney, gut, spleen, skin, and liver. Chemically synthesized QS-CATH exhibited pronounced antibacterial activity against Shigella flexneri, Staphylococcus warneri, Escherichia coli, Salmonella enterica, and Listeria monocytogenes. Furthermore, QS-CATH disrupted the cell membrane integrity of S. flexneri, as evidenced by a lactate dehydrogenase release assay, and it hydrolyzed the genomic DNA of S. flexneri. Additionally, QS-CATH elicited chemotaxis and modulated the expression of inflammatory cytokine genes in RAW264.7 mouse leukemic monocyte/macrophage cells. These findings confirm the antimicrobial effects of amphibian cathelicidin and its ability to influence immune cell function. This will expedite the potential utilization of amphibian antimicrobial peptides as therapeutic agents.
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Anuros , Catelicidinas , Animais , Camundongos , Sequência de Aminoácidos , Fatores Imunológicos/farmacologia , Aeromonas hydrophila , Proteínas de Anfíbios/farmacologia , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/isolamento & purificação , Anti-Infecciosos/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Células RAW 264.7 , Agentes de Imunomodulação/farmacologia , Agentes de Imunomodulação/isolamento & purificação , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/imunologia , População do Leste AsiáticoRESUMO
Cyclic peptides are polypeptide chains formed by cyclic sequences of amide bonds between protein-derived or non-protein-derived amino acids. Compared to linear peptides, cyclic peptides offer several unique advantages, such as increased stability, stronger affinity, improved selectivity, and reduced toxicity. Cyclic peptide has been proved to have a promising application prospect in the medical field. In addition, this paper mainly describes that cyclic peptides play an important role in anti-cancer, anti-inflammatory, anti-virus, treatment of multiple sclerosis and membranous nephropathy through immunomodulation. In order to know more useful information about cyclic peptides in clinical research and drug application, this paper also summarizes cyclic peptides currently in the clinical trial stage and cyclic peptide drugs approved for marketing in the recent five years. Cyclic peptides have many advantages and great potential in treating various diseases, but there are still many challenges to be solved in the development process of cyclic peptides.
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Neuropathic pain has a complex pathogenesis. Here, we examined the role of caveolin-1 (Cav-1) in the anterior cingulate cortex (ACC) in a chronic constriction injury (CCI) mouse model for the enhancement of presynaptic glutamate release in chronic neuropathic pain. Cav-1 was localized in glutamatergic neurons and showed higher expression in the ACC of CCI versus sham mice. Moreover, the release of glutamate from the ACC of the CCI mice was greater than that of the sham mice. Inhibition of Cav-1 by siRNAs greatly reduced the release of glutamate of ACC, while its overexpression (induced by injecting Lenti-Cav-1) reversed this process. The chemogenetics method was then used to activate or inhibit glutamatergic neurons in the ACC area. After 21 days of injection of AAV-hM3Dq in the sham mice, the release of glutamate was increased, the paw withdrawal latency was shortened, and expression of Cav-1 in the ACC was upregulated after intraperitoneal injection of 2 mg/kg clozapine N-oxide. Injection of AAV-hM4Di in the ACC of CCI mice led to the opposite effects. Furthermore, decreasing Cav-1 in the ACC in sham mice injected with rAAV-hM3DGq did not increase glutamate release. These findings suggest that Cav-1 in the ACC is essential for enhancing glutamate release in neuropathic pain.
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Ácido Glutâmico , Neuralgia , Animais , Camundongos , Caveolina 1/genética , Caveolina 1/metabolismo , Ácido Glutâmico/metabolismo , Giro do Cíngulo/metabolismo , Neuralgia/metabolismo , Neuralgia/patologia , Neurônios/patologiaRESUMO
Brevinins exhibit a wide range of structural features and strong biological activities. Brevinin-2, derived from several amphibians, has shown antimicrobial activities. However, little is known about the wound-healing activity of brevinin-2. In this study, brevinin-2 cDNA was identified from the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus) and it comprises a signal peptide, a propeptide, and a mature peptide. Sequence alignment with brevinin-2 derived from other amphibians showed variability of the mature peptide, and the presence of a C-terminal cyclic heptapeptide domain (Cys-Lys-Xaa4-Cys) in the mature peptide. Dark-spotted frog brevinin-2 belonged to the brevinin-2 cluster and was closely related to brevinin-2HB1 from Pelophylax hubeiensis. Synthetic dark-spotted frog brevinin-2 mature peptide (brevinin-2PN) exhibited antibacterial activity against several pathogens by destroying cell membrane integrity and hydrolysis of genomic DNA. Brevinin-2PN exhibited significant wound-healing activity by accelerating the healing of human skin fibroblast cell scratches, influencing cell migration, and stimulating gene expression of growth factors.
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Proteínas de Anfíbios , Peptídeos Antimicrobianos , Sequência de Aminoácidos , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Anuros/genética , DNA Complementar/metabolismo , Humanos , Sinais Direcionadores de Proteínas , Ranidae/genética , Pele/metabolismoRESUMO
The ß-defensins are important components of the vertebrate innate immune system. While mammalian ß-defensins have wide-ranging antibacterial and immunomodulatory activities, those of amphibians remain largely uncharacterised. In this study, ß-defensin cDNA was identified from the skin transcriptome of the Chinese spiny frog Quasipaa spinosa. This ß-defensin (QS-BD) consists of a signal and a mature peptide. Sequence alignments with other amphibian ß-defensins showed conservation of the functional mature peptide and that its closest relative is ß-defensin from Zhangixalus puerensis. Synthetic QS-BD showed antibacterial activity against Vibrio vulnificus, Vibrio harveyi, Streptococcus iniae, and Aeromonas hydrophila. QS-BD showed bactericidal activity by destroying the cell membrane integrity, but did not hydrolyse genomic DNA. QS-BD treatment promoted respiratory bursts and upregulated the expression of interleukin-1ß and tumour necrosis factor-α in the murine leukemic monocyte/macrophage cell line RAW264.7. This is the first demonstration of immunomodulatory activity by an amphibian ß-defensin.
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beta-Defensinas , Animais , Antibacterianos/metabolismo , Anuros/metabolismo , China , Mamíferos , Camundongos , Ranidae/genética , beta-Defensinas/genética , beta-Defensinas/metabolismoRESUMO
Cathelicidins are an important antimicrobial peptide family and are expressed in many different vertebrates. They play an important role in the innate immune system of the host. However, amphibian cathelicidins are poorly understood. In this study, the cDNA of the cathelicidin gene was obtained from the skin transcriptome of tiger frog (Hoplobatrachus rugulosus). The predicted amino acid sequence of tiger frog cathelicidin (HR-CATH) comprises a signal peptide, a cathelin domain, and a mature peptide. The HR-CATH amino acid sequence alignment with other frog cathelicidins showed that the functional mature peptide is highly variable in amphibians, whereas the cathelin domain is conserved. A phylogenetic tree analysis showed that HR-CATH is most closely related to cathelicidin-NV from Nanorana ventripunctata. HR-CATH was chemically synthesized and its in vitro activity was determined. It had high antibacterial activity against Vibrio parahaemolyticus, Staphylococcus aureus, and the pathogenic bacterium Aeromonas hydrophila. HR-CATH damaged the cell membrane integrity of A. hydrophila according to a lactate dehydrogenase release assay and was able to hydrolyze the genomic DNA from A. hydrophila in a dose-dependent manner. Furthermore, in RAW264.7 cells (mouse leukemic monocyte/macrophage cell line), HR-CATH induced chemotaxis and enhanced respiratory burst. Our study shows that amphibian cathelicidin has antimicrobial activity and an immunomodulatory effect on immune cells.
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Proteínas de Anfíbios , Antibacterianos , Anuros/imunologia , Bactérias/efeitos dos fármacos , Catelicidinas , Proteínas de Anfíbios/imunologia , Proteínas de Anfíbios/farmacologia , Animais , Antibacterianos/imunologia , Antibacterianos/farmacologia , Catelicidinas/imunologia , Catelicidinas/farmacologia , Camundongos , Células RAW 264.7RESUMO
NK-lysins (NKLs) are a family of multifunctional antimicrobial peptides that have activity against various microorganisms. However, the immunomodulatory activity of NKL in fish remains unclear. In this study, the cDNA sequence of barbel steed (Hemibarbus labeo) NKL gene was cloned. Barbel steed NKL amino acid sequence comprised a signal peptide and a mature peptide. The saposin B domain in the mature peptide has six conserved cysteines that form three disulfide bonds. Phylogenetic analysis showed that the barbel steed NKL was most closely related to that of the common carp (Cyprinus carpio) NKL. Differential expression analysis showed that the barbel steed NKL gene was expressed in all tested tissues, with the highest expression in the spleen. In response to Aeromonas hydrophila infection, NKL was significantly upregulated in the liver, spleen, head kidney, and gill. The barbel steed NKL showed strong antibacterial activity against Vibrio parahaemolyticus, V. alginolyticus, V. vulnificus, and Listeria monocytogenes. However, NKL had no antibacterial activity against the pathogenic bacteria A. hydrophila. Lactate dehydrogenase release assays showed that NKL damaged the V. parahaemolyticus cell membrane. NKL significantly increased barbel steed survival rate after A. hydrophila infection and upregulated IL-1ß and TNF-α expression in the spleen and head kidney. NKL induced monocyte/macrophage chemotaxis and enhanced the respiratory burst and proinflammatory cytokine expression. Our study shows that fish NKL exhibits immunomodulatory effects and protects the host from pathogenic infections independent of direct bacterial clearance.
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Aeromonas hydrophila/imunologia , Carpas/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Proteolipídeos/imunologia , Sequência de Aminoácidos/genética , Animais , Carpas/microbiologia , Membrana Celular/patologia , Quimiotaxia/imunologia , Clonagem Molecular , Infecções por Bactérias Gram-Negativas/prevenção & controle , Rim Cefálico/metabolismo , Imunomodulação/imunologia , Interleucina-1beta/metabolismo , Listeria monocytogenes/imunologia , Domínios Proteicos/genética , Proteolipídeos/genética , Baço/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vibrio/imunologiaRESUMO
We reported the complete mitochondrial genome (mitogenome) of broad-folded frog (Hylarana latouchii). This mitogenome is 17,007 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and one non-coding sequence (D-loop). The total composition was 58.54% A + T and 41.46% G + C (T: 29.31%, C: 27.33%, A: 29.23%, and G: 14.13%). The phylogenetic analysis revealed that H. latouchii formed a clade with other two species of genus Hylarana. This mitogenomic sequence of H. latouchii provides useful data to study its population genetics and phylogeography.
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The complete mitochondrial DNA (mtDNA) for Pareas stanleyi was determined in this study. The length of complete mtDNA was 18,932 bp, including 13 protein-coding genes (PCGs) (COI-III, ND1-6, ND4L, ATP6, ATP8 and CYTB), 23 tRNA genes, 2 rRNA genes, a L-chain replication-initiating non-coding region (NCR) and 2 control regions. The overall base composition of the sequence is 24.76% of T, 29.20% of C, 30.87% of A, and 15.16% of G, with a total A + T content of 55.63%. The phylogenetic tree showed that P. stanleyi had a close relationship with the other two species (P. boulengeri and P. formosensis) from the genus Pareas.
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The complete mitochondrial DNA (mtDNA) for Odorrana exiliversabilisLi, Ye and Fei 2001 (Anura: Ranidae) was determined in this study. The length of complete mtDNA was 17,122 bp, including 13 PCGs (COI-III, ND1-6, ND4L, ATP6, ATP8 and CYTB), 25 tRNA genes, 2 rRNA genes, 2 non-coding regions of a L-strand replication origin and a control region. The overall base composition of the sequence is 28.27% T, 28.27% C, 28.52% A, and 14.94% G, with a total A + T content of 56.79%. The phylogenetic tree showed that O. exiliversabilis was the sister species of O. tormota, and formed a monophyletic clade with other Odorrana species. These data provide a powerful tool for evolutionary biology and population genetics of genus Odorrana.
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The black-spotted stout newt (Pachytriton brevipes) is widely distributed in the mountains of southeastern China. In this study, we sequenced and determined the complete mitochondrial genome (mitogenomes) of two P. brevipes samples collected in Mount Wuyi. The assembled mitogenomes were 16,298 bp and 16,301 bp in length, and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, one non-coding region, and one control region. The phylogenetic analysis indicated the two samples of black-spotted stout newt grouped together and are the sister group of P. feii.
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We report the complete mitochondrial genome (mtDNA) of Pareas formosensis (Squamata: Colubridae). This circular mtDNA is 17,703 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and 2 non-coding sequence (D-loop). The total of mtDNA was composition of 57.26% A + T and 42.74% G + C (T: 25.21%, C: 28.84%, A: 32.05%, G: 13.90%). The phylogenetic analysis revealed that P. formosensis formed a clade with other species of Pareas. This mtDNA sequence of P. formosensis provides useful data for studying the population genetics and phylogeography of Colubridae.
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Paramesotriton aurantius (Caudata: Salamandridae) is a new species that found in southeastern China. Its complete mitochondrial genome (mitogenome) sequence was 16,313 bp in length with with A + T contents of 60.9%, and contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, one control region (D-loop), and one non-coding region. Our molecular tree showed that P. aurantius was positioned near P. hongkongensis, and formed a clade with other Paramesotriton species. The first complete mitogenome sequence of P. aurantius could provided fundamental data for resolving phylogenetic and genetic problems related to genus Paramesotriton.
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OBJECTIVE: To construct the HPV16 L1 prokaryotic expression plasmid and to optimize its expression. METHODS: A pair of primers was designed according to plasmid sequences of pGEX-KG and the HPV16L1 genes published by GeneBank. The DNA fragment of 1500 bp was amplified by PCR from the HPV recombinant plasmid with HPV16L1 gene, then cloned into pGEX-KG and transformed into the host E.coli strain JM109. The pGEX-KG-HPV16L1 plasmid was taken and transformed into BL21(DE3) for expression. Induced by IPTG at 37 degree, the expression product of HPV16L1 gene was identified by SDS-PAGE and Western blot. RESULTS: HPV16L1 fusion protein was expressed successfully in the form of inclusion bodies. The molecular weight was 83 kD. Meanwhile, the optimum condition of HPV16L1 fusion protein expression was induced with 1.0 mmol*L(-1) IPTG for 4 h. The fusion protein reacted specifically with antibodies against HPV16L1. CONCLUSION: The prokaryotic expression vector of HPV16L1 gene has been constructed and expressed in E.coli successfully.