Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments

The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca²⁺, in vitro. Analysis of a mutant that bears a point mutation at the Ca²⁺ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca²⁺ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.     

Berberis libanotica Ehrenb Extract Shows Anti-Neoplastic Effects on Prostate Cancer Stem/Progenitor Cells

Cancer stem cells (CSCs), including those of advanced prostate cancer, are a suggested reason for tumor resistance toward conventional tumor therapy. Therefore, new therapeutic agents are urgently needed for targeting CSCs. Despite the minimal understanding of their modes of action, natural products and herbal therapies have been commonly used in the prevention and treatment of many cancers. Berberis libanotica Ehrenb (BLE) is a plant rich in alkaloids which may possess anti-cancer activity and a high potential for eliminating CSCs. We tested the effect of BLE on prostate cancer cells and our data indicated that this extract induced significant reduction in cell viability and inhibited the proliferation of human prostate cancer cell lines (DU145, PC3 and 22Rv1) in a dose- and time-dependent manner. BLE extract induced a perturbation of the cell cycle, leading to a G0-G1 arrest. Furthermore, we noted 50% cell death, characterized by the production of high levels of reactive oxidative species (ROS). Inhibition of cellular migration and invasion was also achieved upon treatment with BLE extract, suggesting a role in inhibiting metastasis. Interestingly, BLE extract had a major effect on CSCs. Cells were grown in a 3D sphere-formation assay to enrich for a population of cancer stem/progenitor cells. Our results showed a significant reduction in sphere formation ability. Three rounds of treatment with BLE extract were sufficient to eradicate the self-renewal ability of highly resistant CSCs. In conclusion, our results suggest a high therapeutic potential of BLE extract in targeting prostate cancer and its CSCs.     

LncRNA Gm2044 highly expresses in spermatocyte and inhibits Utf1 translation by interacting with Utf1 mRNA

Spermatogenesis is a process which includes the following phases: spermatogonial stem cell proliferation and differentiation, spermatogonia, spermatocyte, spermatid and mature sperm. Spermatogenic failure is the important factor resulting in male infertility. Recent studies showed that long noncoding RNA (lncRNA) have been found to be involved in the regulation of male reproduction. However, lncRNA associated with spermatogenesis and their mechanisms of action are unclear. The aim of this study is to explore the role and molecular mechanism of lncRNA in spermatogenesis. LncRNA microarray of germ cells and bioinformatic analysis showed lncRNA Gm2044 may play potential roles in spermatogenesis. The expression level of RNA and protein were analyzed by RT-qPCR and western blotting, respectively. The interaction of lncRNA with mRNA was detected by RNA pull down and cellular proliferation was measured using CCK-8 reagent. Testis-enriched lncRNA Gm2044 is abundant in mouse spermatocytes. Gm2044 can suppress the translation of adjacent spermatogenesis-related gene Utf1 by interacting with Utf1 mRNA. Furthermore, the proliferation of mouse spermatogonia GC-1 cell line and spermatocyte GC-2 cell line was inhibited by Gm2044. CONCLUSION: LncRNA Gm2044 was identified to inhibit Utf1 mRNA translation and play important roles in spermatogenesis.     

MicroRNA‐130b targets PTEN to induce resistance to cisplatin in lung cancer cells by activating Wnt/β‐catenin pathway

More and more studies indicate the relevance of miRNAs in inducing certain drug resistance. Our study aimed to investigate whether microRNA‐130b‐3p (miR‐130b) mediates the chemoresistance as well as proliferation of lung cancer (LC) cells. MTS assay and apoptosis analysis were conducted to determine cell proliferation and apoptosis, respectively. Binding sites were identified using a luciferase reporter system, whereas mRNA and protein expression of target genes was determined by RT‐PCR and immunoblot, respectively. Mouse xenograft model was used to evaluate the role of miR‐130b in cisplatin resistance in vivo. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR ) versus its parental cell lines, indicated its crucial relevance for LC biology. We identified PTEN as miR‐130b's major target and inversely correlated with miR‐130b expression in LC. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. Suppression of miR‐130b enhanced drug cytotoxicity and reduced proliferation of A549/CR cells both internally and externally. Particularly, miR‐130b mediated Wnt/β‐catenin signalling pathway activities, chemoresistance and proliferation in LC cell, which was partially blocked following knockdown of PTEN. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway. The rising level of miR‐130b in cisplatin resistance LC cell lines (A549/CR and H446/CR) versus its parental cell lines, indicated its crucial relevance for LC biology. Moreover, excessive miR‐130b expression promoted drug resistance and proliferation, decreased apoptosis of A549 cells. These findings suggest that miR‐130b targets PTEN to mediate chemoresistance, proliferation, and apoptosis via Wnt/β‐catenin pathway.     

Purification and Conformational Characterization of a Novel Interleukin-2 Mutein

The Protein Journal - Toxicity of high-dose IL-2-based therapies have motivated the development of the IL-2 mutein, which has low expansion properties for regulatory T lymphocytes. The development...     

Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family

A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.     

Biphasic effect of extra-reticular Mg2+ on hepatic G6P transport and hydrolysis

Magnesium ions (Mg²⁺) play a key role in regulating hepatic cellular functions and enzymatic activities. In the present study, we report a concentration-dependent effect of cytosolic Mg²⁺ on G6P and pyrophosphate (PPi) transport and hydrolysis in digitonin-permeabilized rat hepatocytes. The stimulatory effect of Mg²⁺ on G6P is specific but biphasic, with a maximal effect at a concentration of 0.25 mM, whereas the effect on PPi increases in a dose-dependent manner. Both effects can be abolished by addition of EDTA to the system. Addition of taurocholate, histone-2A, alamethicin or A23187 to the incubation system results in a marked decrease in the Mg²⁺ concentration present within the endoplasmic reticulum lumen. Under these conditions, the stimulatory effect of extra-reticular Mg²⁺ on G6P transport and hydrolysis is abolished. Taken together, these data suggest that cytosolic Mg²⁺ stimulates G6P transport by acting at the level of the substrate binding site of the G6Pase enzymatic complex or the surrounding phospholipid environment. The effect, which is lost when G6P has readily access to the ER lumen, requires physiological endoplasmic reticulum Mg²⁺ content.