Expression and localization of forkhead transcriptional factor 2 (Foxl2) in the gonads of protogynous wrasse, Halichoeres trimaculatus

<p>Abstract</p> <p>Background</p> <p>Three-spot wrasse, <it>Halichoeres trimaculatus</it>, is a marine protogynous hermaphrodite fish. Individuals mature either as initial phase (IP) males or females. Appropriate social cues induce the sex change from IP female to terminal phase (TP) male. However, the molecular mechanisms behind such a sex change remain largely unknown. Recently, the forkhead transcription factor 2 (Foxl2) was identified as an essential regulator of vertebrate ovarian development/function/phenotype. Inspired by this information, we characterized the expression patterns of Foxl2 in the protogynous wrasse assuming Foxl2 as the female-specific marker in this species.</p> <p>Methods</p> <p>First, we clonedFoxl2 cDNA from ovary by reverse transcription polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). Next, we analysed expression pattern of Foxl2 messenger RNA (mRNA) and protein in gonads of different sexual phases by real time quantitative PCR assay and flour fluorescence immunohistochemical method, respectively. Additionally, we studied the changes in Foxl2 expression pattern during aromatase inhibitor (AI)-induced sex change.</p> <p>Results</p> <p>The amino acid sequence (306 AA) of wrasse Foxl2, especially the forkhead domain, shows high identity with that of other reported teleost Foxl2s. Quite unexpectedly, no sexual dimorphism was observable between the testes and ovary in the expression pattern of Foxl2. In female phase fish, signals for Foxl2 protein were detectable in the granulosa cells, but not the theca cells. Transcript levels of Foxl2 in the testes of IP and TP males were identical to that in the ovaries of females and, further, Foxl2 protein was found to be localized in the interstitial cells including tubules and Leydig cells. Treatment with AI induced sex change in male gonads and an up-regulation was seen in the expression of Foxl2 in these gonads.</p> <p>Conclusions</p> <p>Unlike in other vertebrates, including teleosts, Foxl2 may have a different role in the naturally sex changing fishes.</p

Identification of α-type subunits of the Xenopus 20S proteasome and analysis of their changes during the meiotic cell cycle

BACKGROUND: The 26S proteasome is the proteolytic machinery of the ubiquitin-dependent proteolytic system responsible for most of the regulated intracellular protein degradation in eukaryotic cells. Previously, we demonstrated meiotic cell cycle dependent phosphorylation of α4 subunit of the 26S proteasome. In this study, we analyzed the changes in the spotting pattern separated by 2-D gel electrophoresis of α subunits during Xenopus oocyte maturation. RESULTS: We identified cDNA for three α-type subunits (α1, α5 and α6) of Xenopus, then prepared antibodies specific for five subunits (α1, α3, α5, α6, and α7). With these antibodies and previously described monoclonal antibodies for subunits α2 and α4, modifications to all α-type subunits of the 26S proteasome during Xenopus meiotic maturation were examined by 2D-PAGE. More than one spot for all subunits except α7 was identified. Immunoblot analysis of 26S proteasomes purified from immature and mature oocytes showed a difference in the blots of α2 and α4, with an additional spot detected in the 26S proteasome from immature oocytes (in G2-phase). CONCLUSIONS: Six of α-type subunits of the Xenopus 26S proteasome are modified in Xenopus immature oocytes and two subunits (α2 and α4) are modified meiotic cell cycle-dependently

Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation

BACKGROUND: During Xenopus oocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48. RESULTS: p48 was purified by conventional column chromatography. The resulting purified fraction contained two other proteins with molecular masses of 30 (p30) and 37 (p37) kDa. cDNAs encode elongation factor-1γ and δ were obtained by an immuno-screening method using polyclonal antibodies against purified p48 complex, which recognized p48 and p37. N-terminal amino acid sequence analysis of p30 revealed that it was identical to EF-1β. To identify the p48 complex bound to the 26S proteasome as EF-1βγδ, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 β,γ and δ were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1γ. Cross-reactivity of the antibodies toward the p48 complex and recombinant proteins showed it to be specific for each component. These results indicate that the p48 complex bound to the 26S proteasome is the EF-1 complex. MPF phosphorylated EF-1γ was shown to bind to the 26S proteasome. When EF-1γ is phosphorylated by MPF, the association is stabilized. CONCLUSION: p48 bound to the 26S proteasome is identified as the EF-1γ. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation

Serum REIC/Dickkopf-3 Protein Level Predicts Disease-Free Survival in Patients with Hepatocellular Carcinoma

The physiological role of the reduced expression of immortalized cells (REIC)/Dickkopf-3 (Dkk-3) protein in patients with hepatocellular carcinoma (HCC) remains unclear. In this study, we evaluated the effect of the REIC/Dkk-3 protein on HCC cell proliferation and assessed the relationship between the serum REIC/Dkk-3 protein level and the prognosis in patients with HCC. We evaluated the REIC/Dkk-3 protein-induced anticancer effects on Huh7 and Hep3B cells (HCC cell lines) in the presence of peripheral blood mononuclear cells (PBMCs), and found that combination treatment with REIC/Dkk-3 protein and PBMCs reduced the proliferation of HCC cells (Hep3B: 82.0%±16.3%; Huh7: 72.6%±9.1%). We also studied 194 HCC patients who underwent primary liver resection or primary radiofrequency ablation from 2008 to 2017. Serum REIC/Dkk-3 protein levels were measured by an enzyme-linked immunosorbent assay and compared to the prognostic data. The 3-year disease-free survival of the REIC/Dkk-3 high group was significantly higher than that in the REIC/Dkk-3 low group. In conclusion, this is the first study investigating the relationship between HCC patient survival and serum REIC/Dkk-3 protein levels in a large population. Based on the results, the serum REIC/Dkk-3 protein level should be considered a new prognostic marker for patients with HCC

Discovery of a kleptoplastic 'dinotom' dinoflagellate and the unique nuclear dynamics of converting kleptoplastids to permanent plastids

A monophyletic group of dinoflagellates, called ‘dinotoms’, are known to possess evolutionarily intermediate plastids derived from diatoms. The diatoms maintain their nuclei, mitochondria, and the endoplasmic reticulum in addition with their plastids, while it has been observed that the host dinoflagellates retain the diatoms permanently by controlling diatom karyokinesis. Previously, we showed that dinotoms have repeatedly replaced their diatoms. Here, we show the process of replacements is at two different evolutionary stages in two closely related dinotoms, Durinskia capensis and D. kwazulunatalensis. We clarify that D. capensis is a kleptoplastic protist keeping its diatoms temporarily, only for two months. On the other hand, D. kwazulunatalensis is able to keep several diatoms permanently and exhibits unique dynamics to maintain the diatom nuclei: the nuclei change their morphologies into a complex string-shape alongside the plastids during interphase and these string-shaped nuclei then condense into multiple round nuclei when the host divides. These dynamics have been observed in other dinotoms that possess permanent diatoms, while they have never been observed in any other eukaryotes. We suggest that the establishment of this unique mechanism might be a critical step for dinotoms to be able to convert kleptoplastids into permanent plastids.info:eu-repo/semantics/publishedVersio

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems

Higher expression levels of SOCS 1,3,4,7 are associated with earlier tumour stage and better clinical outcome in human breast cancer

Background Suppressors of cytokine signaling (SOCS) are important negative feedback regulators of the JAK/STAT signaling pathway, and have been recently investigated for their role in the development of different cancers. In this study, we examined the expression of SOCS1-7 genes in normal and breast cancer tissue and correlated this with several clinico-pathological and prognostic factors. Methods SOCS1-7 mRNA extraction and reverse transcription were performed on fresh frozen breast cancer tissue samples (n = 127) and normal background breast tissue (n = 31). Transcript levels of expression were determined using real-time PCR and analyzed against TNM stage, tumour grade and clinical outcome over a 10 year follow-up period. Results SOCS1,4,5,6 and 7 expression decreased with increased TNM stage (TNM1 vs. TNM3 p = 0.039, TNM1 vs. TNM4 p = 0.016, TNM2 vs. TNM4 p = 0.025, TNM1 vs. TNM3 p = 0.012, and TNM1 vs. TNM3 p = 0.044 respectively). SOCS2 and 3 expression decreased with increased Nottingham Prognostic Index (NPI) (NPI1 vs. NPI3 p = 0.033, and NPI2 vs. NPI3 p = 0.041 respectively). SOCS7 expression decreased with higher tumour grade (Grade 3 vs. Grade 2 p = 0.037). After a median follow up period of 10 years, we found higher levels of SOCS1,2 and 7 expression among those patients who remained disease-free compared to those who developed local recurrence (p = 0.0073, p = 0.021, and p = 0.039 respectively). Similarly, we found higher levels of SOCS 2,4, and 7 expression in those who remained disease-free compared to those who developed distant recurrence (p = 0.022, p = 0.024, and p = 0.033 respectively). Patients who remained disease-free had higher levels of SOCS1 and 2 expression compared to those who died from breast cancer (p = 0.02 and p = 0.033 respectively). The disease free survival (DFS) and overall survival (OS) curves showed that higher levels of SOCS1, 3 and 7 were significant predictors of higher DFS (p = 0.015, p = 0.024 and 0.03 respectively) and OS (p = 0.005, p = 0.013 and p = 0.035 respectively). Higher levels of SOCS 4 were significant in predicting better OS (p = 0.007) but not DFS. Immunohistochemical staining of representative samples showed a correlation between SOCS1, 3, 7 protein staining and the SOCS1, 3, 7 mRNA expression. Conclusion Higher mRNA expression levels of SOCS1, 3, 4 and 7 are significantly associated with earlier tumour stage and better clinical outcome in human breast cancer

Kleptochloroplast Enlargement, Karyoklepty and the Distribution of the Cryptomonad Nucleus in Nusuttodinium (= Gymnodinium) aeruginosum (Dinophyceae)

The unarmoured freshwater dinoflagellate Nusuttodinium (= Gymnodinium) aeruginosum retains a cryptomonad-derived kleptochloroplast and nucleus, the former of which fills the bulk of its cell volume. The paucity of studies following morphological changes to the kleptochloroplast with time make it unclear how the kleptochloroplast enlarges and why the cell ultimately loses the cryptomonad nucleus. We observed, both at the light and electron microscope level, morphological changes to the kleptochloroplast incurred by the enlargement process under culture conditions. The distribution of the cryptomonad nucleus after host cell division was also investigated. The volume of the kleptochloroplast increased more than 20-fold, within 120 h of ingestion of the cryptomonad. Host cell division was not preceded by cryptomonad karyokinesis so that only one of the daughter cells inherited a cryptomonad nucleus. The fate of all daughter cells originating from a single cell through five generations was closely monitored, and this observation revealed that the cell that inherited the cryptomonad nucleus consistently possessed the largest kleptochloroplast for that generation. Therefore, this study suggests that some important cryptomonad nucleus division mechanism is lost during ingestion process, and that the cryptomonad nucleus carries important information for the enlargement of the kleptochloroplast