RESUMO
Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.
Assuntos
Anticoagulantes , Proteínas de Artrópodes , Sulfotransferases , Carrapatos , Animais , Anticoagulantes/química , Sulfotransferases/química , Tirosina/metabolismo , Proteínas de Artrópodes/química , CristalizaçãoRESUMO
Osteosarcoma is a malignant tumor that produces neoplastic bone or osteoid osteoma. In human multicentric osteosarcoma (HMOS), a unique variant of human osteosarcoma (HOS), multiple bone lesions occur simultaneously or asynchronously before lung metastasis. HMOS is associated with an extremely poor prognosis, and effective treatment options are lacking. Using the proteins in our previously generated HMOS cell lines as antigens, we generated antibodies using a human antibody phage library. We obtained antibody clones recognizing 95 independent antigens and developed a fluorescence probe-based enzyme-linked immunosorbent assay (ELISA) technique capable of evaluating the reactivity of these antibodies by fluorescence intensity, allowing simple, rapid, and high-throughput selection of antibody clones. These results were highly correlated with those using flow cytometry. Subsequently, the HMOS cell lysate was incubated with the antibody, the antigen-antibody complex was recovered with magnetic beads, and the protein bands from electrophoresis were analyzed using liquid chromatography-mass spectrometry (LC/MS). CAVIN1/polymerase I transcript release factor was specifically detected in the HMOS cells. In conclusion, we found via a novel high-throughput screening method that CAVIN1/PTRF is an HMOS-specific cell membrane biomarker and an antigen capable of producing human antibodies. In the future, antibody-drug conjugate targeting of these specific proteins may be promising for clinical applications.
RESUMO
The freezing tolerance of plants that live in cold regions increases after exposure to low temperature, a process termed cold acclimation (CA). During CA, restructuring of the plasma membrane (PM) is important to enhance freezing tolerance. We have previously shown that the function of DYNAMIN-RELATED PROTEIN 1 E (DRP1E), which regulates endocytosis by pinching vesicles from the PM, is associated with the enhancement of freezing tolerance during CA in Arabidopsis. DRP1E is predicted to play a role in reconstituting the PM composition during CA. In this study, to test the validity of this hypothesis, we studied the changes in PM proteome patterns induced by drp1e mutation. In a detailed physiological analysis, after 3 days of CA, only young leaves showed significantly less increase in freezing tolerance in the mutant than in the wild type (WT). Using nano-liquid chromatography-tandem mass spectrometry, 496 PM proteins were identified. Among these proteins, 81 or 71 proteins were specifically altered in the WT or the mutant, respectively, in response to CA. Principal component analysis showed that the proteomic pattern differed between the WT and the mutant upon cold acclimation (CA), suggesting that DRP1E contributes to reconstruction of the PM during CA. Cluster analysis revealed that proteins that were significantly increased in the mutant after CA were biased toward glycosylphosphatidylinositol-anchored proteins, such as fasciclin-like arabinogalactan proteins. Thus, a primary target of DRP1E-associated PM reconstruction during CA is considered to be glycosylphosphatidylinositol-anchored proteins, which may be removed from the PM by DRP1E in young leaves after 3 days of CA.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Congelamento , Proteômica/métodos , Glicosilfosfatidilinositóis/metabolismo , Aclimatação/fisiologia , Membrana Celular/metabolismo , Temperatura Baixa , Dinaminas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMO
Despite being one of the bilaterians, the body plan of echinoderms shifts from bilateral symmetry to five-fold radial, or pentaradial symmetry during embryogenesis or their metamorphosis. While the clarification of the developmental mechanism behind this transition will be a basis for understanding their unique body plan evolution, it is still poorly understood. With this regard, the hydrocoel, a mesodermal coelom formed on the left side of bilateral larva, would be a clue for understanding the mechanism as it is the first pentaradial structure that appears before metamorphosis and develops into the water vascular system of adults. By analyzing the development of a sea cucumber, Apostichopus japonicus, we found that the hydrocoel expresses genes related in muscle and neural formation such as myosin heavy chain, tropomyosin, soxC, and elav, implying that cells of the hydrocoel contributes to muscle and neural structures in the adult. Furthermore, ablation of one of the hydrocoel lobes led to incomplete development of adult pentameral structures. The ablation of primary hydrocoel lobes resulted in the reduction in tentacles and the ablation of secondary hydrocoel lobes resulted in the reduction in water vascular canals and nerve cords. Our findings suggest that the hydrocoel lobes may serve as a potential organizing center for establishing the pentaradial body plan in echinoderms.
Assuntos
Pepinos-do-Mar , Stichopus , Animais , Metamorfose Biológica/fisiologia , Equinodermos , ÁguaRESUMO
To clarify the acoustic variables for predicting and classifying Japanese singleton and geminate consonants, raw and logarithmic durations of the consonants and their related segments were examined using 12 minimal pair words that were pronounced in a carrier sentence at various speaking rates by 20 native Japanese speakers. Regression and discriminant analyses revealed that the logarithmic durations were better at predicting and classifying Japanese singleton and geminate consonants than the raw durations used in many previous studies. Specifically, the best acoustic variables were the logarithmic duration of the consonant's closure or frication and the logarithmic average duration of the mora in the preceding carrier phrase. These results suggest that logarithmic durations are relational invariant acoustic variables that can cope with the durational variations of singleton and geminate consonants in a wide range of speaking rates.
Assuntos
Fonética , Acústica da Fala , Acústica , Japão , IdiomaRESUMO
Echinoderms are an exceptional group of bilaterians that develop pentameral adult symmetry from a bilaterally symmetric larva. However, the genetic basis in evolution and development of this unique transformation remains to be clarified. Here we report newly sequenced genomes, developmental transcriptomes, and proteomes of diverse echinoderms including the green sea urchin (L. variegatus), a sea cucumber (A. japonicus), and with particular emphasis on a sister group of the earliest-diverged echinoderms, the feather star (A. japonica). We learned that the last common ancestor of echinoderms retained a well-organized Hox cluster reminiscent of the hemichordate, and had gene sets involved in endoskeleton development. Further, unlike in other animal groups, the most conserved developmental stages were not at the body plan establishing phase, and genes normally involved in bilaterality appear to function in pentameric axis development. These results enhance our understanding of the divergence of protostomes and deuterostomes almost 500 Mya.
Assuntos
Equinodermos/genética , Lytechinus/genética , Stichopus/genética , Exoesqueleto/anatomia & histologia , Animais , Evolução Biológica , DNA/genética , Equinodermos/anatomia & histologia , Equinodermos/embriologia , Equinodermos/crescimento & desenvolvimento , Biblioteca Gênica , Genes Homeobox/genética , Genoma/genética , Lytechinus/anatomia & histologia , Lytechinus/crescimento & desenvolvimento , Filogenia , Proteômica , Análise de Sequência de DNA , Stichopus/anatomia & histologia , Stichopus/crescimento & desenvolvimentoRESUMO
In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.
Assuntos
Endoderma/fisiologia , Proteínas de Homeodomínio/fisiologia , Mesoderma/fisiologia , Ouriços-do-Mar/embriologia , Animais , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Larva/fisiologia , Fenótipo , Filogenia , Receptores Notch/fisiologia , Ouriços-do-Mar/genéticaRESUMO
Sea cucumbers (a class of echinoderms) exhibit a high capacity for regeneration, such that, following ejection of inner organs in a process called evisceration, the lost organs regenerate. There are two ways by which evisceration occurs in sea cucmber species: from the mouth (anterior) or the anus (posterior). Intriguingly, regenerating tissues are formed at both the anterior and posterior regions and extend toward the opposite ends, and merge to form a complete digestive tract. From the posterior side, the digestive tube regenerates extending a continuous tube from the cloaca, which remains at evisceration. In posteriorly-eviscerating species, the esophagus remains in the body, and a new tube regenerates continuously from it. However, in anterior-eviscerating species, no tubular tissue remains in the anterior region, raising the question of how the new digestive tube forms in the anterior regenerate. We addressed this question by detailed histological observations of the regenerating anterior digestive tract in a small sea cucumber, Eupentacta quinquesemita ("ishiko" in Japanese) after induced-evisceration. We found that an initial rudiment consisting of mesenchymal cells is formed along the edge of the anterior mesentery from the anterior end, and then, among the mesenchymal cells, multiple clusters of epithelial-like cells appears simultaneously and repeatedly in the extending region by mesenchymal-epithelial transition (MET) as visulalized using toluidine blue staining. Subsequently, multiple cavities were formed surrounded with these epithelial cells, and appeared to coalesce with each other to form into multiple lumens, and to eventually become a single tube. This anterior tube then fused to the tube regenerated from the posterior rudiment. Thus, we elucidated the process of regeneration of the anterior portion of the gut in an anteriorly eviscerating species, and suggest the involvement of MET and fusion of cavities/lumens in regeneration of the digestive tube.
RESUMO
hox genes are found as clusters in the genome in most bilaterians. The order of genes in the cluster is supposed to be correlated with the site of expression along the anterior-posterior body axis and the timing of expression during development, and these correlations are called spatial and temporal collinearity, respectively. Here we studied the expression dynamics of all hox genes of the diploid species Xenopus tropicalis in four Hox clusters (A-D) by analyzing high-temporal-resolution RNA-seq databases and the results showed that temporal collinearity is not supported, which is consistent with our previous data from allotetraploid X enopus laevis Because the temporal collinearity hypothesis implicitly assumes the collinear order of gene activation, not mRNA accumulation, we determined for the first time the timing of when new transcripts of hox genes are produced, by detecting pre-spliced RNA in whole embryos with reverse transcription and quantitative PCR (RT-qPCR) for all hoxa genes as well as several selected hoxb, hox c and hoxd genes. Our analyses showed that, coinciding with the RNA-seq results, hoxa genes started to be transcribed in a non-sequential order, and found that multiple genes start expression almost simultaneously or more posterior genes could be expressed earlier than anterior ones. This tendency was also found in hoxb and hoxc genes. These results suggest that temporal collinearity of hox genes is not held during early development of Xenopus.
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This study examines the perception of Mandarin lexical tones by native speakers of Burmese who use lexical tones in their first language (L1) but are naïve to Mandarin. Unlike Mandarin tones, which are primarily cued by pitch, Burmese tones are cued by phonation type as well as pitch. The question of interest is whether Burmese listeners can utilize their L1 experience in processing unfamiliar Mandarin tones. Burmese listeners' discrimination accuracy was compared with that of Mandarin listeners and Australian English listeners. The Australian English group was included as a control group with a non-tonal background. Accuracy of perception of six tone pairs (T1-T2, T1-T3, T1-T4, T2-T3, T2-T4, T3-T4) was assessed in a discrimination test. Our main findings are 1) Mandarin listeners were more accurate than non-native listeners in discriminating all tone pairs, 2) Australian English listeners naïve to Mandarin were more accurate than similarly naïve Burmese listeners in discriminating all tone pairs except for T2-T4, and 3) Burmese listeners had the greatest trouble discriminating T2-T3 and T1-T2. Taken together, the results suggest that merely possessing lexical tones in L1 may not necessarily facilitate the perception of non-native tones, and that the active use of phonation type in encoding L1 tones may have played a role in Burmese listeners' less than optimal perception of Mandarin tones.
Assuntos
Povo Asiático/psicologia , Multilinguismo , Fonética , Percepção da Fala , Adulto , Feminino , Humanos , Idioma , Masculino , Mianmar , Percepção da Altura SonoraRESUMO
Sea cucumbers, one main class of Echinoderms, have a very fast and drastic metamorphosis process during their development. However, the molecular basis under this process remains largely unknown. Here we systematically examined the gene expression profiles of Japanese common sea cucumber (Apostichopus japonicus) for the first time by RNA sequencing across 16 developmental time points from fertilized egg to juvenile stage. Based on the weighted gene co-expression network analysis (WGCNA), we identified 21 modules. Among them, MEdarkmagenta was highly expressed and correlated with the early metamorphosis process from late auricularia to doliolaria larva. Furthermore, gene enrichment and differentially expressed gene analysis identified several genes in the module that may play key roles in the metamorphosis process. Our results not only provide a molecular basis for experimentally studying the development and morphological complexity of sea cucumber, but also lay a foundation for improving its emergence rate.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes e Vias Metabólicas/fisiologia , Metamorfose Biológica/fisiologia , Modelos Biológicos , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Pepinos-do-Mar/fisiologia , Animais , Simulação por Computador , Perfilação da Expressão Gênica/métodosRESUMO
From whole genome sequencing of an allotetraploid frog, Xenopus laevis, two homeologous sets (L and S) of four Hox clusters A through D (HoxA.L/S, HoxB.L/S, HoxC.L/S, and HoxD.L/S) and 13 paralogous groups (PGs) with 76 genes were identified, allowing us to carry out the first comprehensive analyses of hox gene expression in vertebrates. Expression of all hox genes during development and in adult tissues was analyzed by RNA-sequencing. The expression levels of most hox genes were similar between homeologs, but in some pairs, large differences were observed and several of these were confirmed by RT-PCR and whole mount in situ hybridization experiments. These results indicate that subfunctionalization of hox genes may have occurred since allotetraploidization. Furthermore, comprehensive analysis of hox gene expression during early development did not agree with the hypothesis of temporal collinearity especially in genes belonging to PG2 to PG10.
Assuntos
Proteínas de Homeodomínio/metabolismo , Animais , Análise por Conglomerados , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox/genética , Genes Homeobox/fisiologia , Proteínas de Homeodomínio/genética , Hibridização In Situ , Xenopus laevisRESUMO
Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.
Assuntos
Genes , Cromossomos Sexuais/genética , Diferenciação Sexual/genética , Xenopus laevis/genética , Animais , Evolução Biológica , Inversão Cromossômica , Elementos de DNA Transponíveis/genética , Diploide , Evolução Molecular , Feminino , Duplicação Gênica , Haploidia , Hibridização in Situ Fluorescente , Masculino , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Processos de Determinação Sexual/genéticaRESUMO
Two siamois-related homeobox genes siamois (sia1) and twin (sia2), have been reported in Xenopus laevis. These genes are expressed in the blastula chordin- and noggin-expressing (BCNE) center and the Nieuwkoop center, and have complete secondary axis-inducing activity when over-expressed on the ventral side of the embryo. Using whole genome sequences of X. tropicalis and X. laevis, we identified two additional siamois-related genes, which are tandemly duplicated near sia1 and sia2 to form the siamois gene cluster. Four siamois genes in X. tropicalis are transcribed at blastula to gastrula stages. In X. laevis, the siamois gene cluster is present on both homeologous chromosomes, XLA3L and XLA3S. Transcripts from seven siamois genes (three on XLA3L and four on XLA3S) in X. laevis were detected at blastula to gastrula stages. A transcribed gene, sia1p. S, encodes an inactive protein without a homeodomain. When over-expressed ventrally, all siamois-related genes tested in this study except for sia1p. S induced a complete secondary axis, indicating that X. tropicalis and X. laevis have four and six active siamois-related genes, respectively. Of note, each gene required different amounts of mRNA for full activity. These results suggest the possibility that siamois cluster genes have functional redundancy to endow robustness and quickness to organizer formation in Xenopus species.
Assuntos
Proteínas de Homeodomínio/genética , Família Multigênica , Proteínas de Xenopus/genética , Xenopus/genética , Sequência de Aminoácidos , Animais , Blástula/metabolismo , Padronização Corporal/genética , Mapeamento Cromossômico , Sequência Conservada , Diploide , Embrião não Mamífero/metabolismo , Gástrula/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Organizadores Embrionários , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Tetraploidia , Via de Sinalização Wnt , Xenopus/embriologia , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
Xenopus laevis has an allotetraploid genome of 3.1Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otx1 suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.