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Abstract  Our previous studies have shown that overexpression of ?1,4-galactosyltransferase1 (?1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of ?1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface ?1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface ?1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of ?1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface ?1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11p58 is a p34cdc2-related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11p58 during postnatal development in mouse testes and examined the cellular localization of CDK11p58 and cyclinD3, which was associated with CDK11p58 in mammalian cells. Western blot analysis revealed that CDK11p58 was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11p58 was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11p58 were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11p58 expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11p58 and cyclinD3 in the adult testeis was revealed by immunofluorescence analysis. (Mol Cell Biochem 270: 99–106, 2005) [ABSTRACT FROM AUTHOR]

Glycosylation is one of the most important post-translational modifications and it is clear that the single step of β-1,4-galactosylation is performed by a family of β-1,4-galactosyltransferases (β4-GalTs) and that each member of this family may play a distinct role in different tissues and cells. In this study, we characterized the gene expression of six β4-GalTs in mouse testis and analyzed the changes of galactosylation of testis glycoproteins during postnatal development. Northern blot analysis revealed that β4-GalT-I and β4-GalT-IV were expressed mainly in newborn mouse testis and that the expression of β4-GalT-II increased markedly and persisted at the highest levels in adult mouse testis. The expression of β4-GalT-III and β4-GalT-V, however, remained relatively at low levels during mouse testicular development. In contrast, the expression of β4-GalT-VI was undetectable in mouse testis. The gene expression of β4-GalT-II in mouse testis was further analyzed by in situ hybridization due to its unique expression pattern. Strong hybridization signals were detected in the seminiferous tubules and the expression varied among the different stages of spermatogenic differentiation. The distinct gene expression patterns of β4-GalTs in mouse testis could affect the differential galactosylation of testis glycoproteins, as revealed by lectin histochemistry analysis. [ABSTRACT FROM AUTHOR]

Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM) may lead to preeclampsia (PE). We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO) annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8) related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE).

In the context of Industry 4.0, the most popular way to identify and track objects is to add tags, and currently most companies still use cheap quick response (QR) tags, which can be positioned by computer vision (CV) technology. In CV, instance segmentation (IS) can detect the position of tags while also segmenting each instance. Currently, the mask region-based convolutional neural network (Mask R-CNN) method is used to realize IS, but the completeness of the instance mask cannot be guaranteed. Furthermore, due to the rich texture of QR tags, low-quality images can lower intersection-over-union (IoU) significantly, disabling it from accurately measuring the completeness of the instance mask. In order to optimize the IoU of the instance mask, a QR tag IS method named the mask UNet region-based convolutional neural network (MU R-CNN) is proposed. We utilize the UNet branch to reduce the impact of low image quality on IoU through texture segmentation. The UNet branch does not depend on the features of the Mask R-CNN branch so its training process can be carried out independently. The pre-trained optimal UNet model can ensure that the loss of MU R-CNN is accurate from the beginning of the end-to-end training. Experimental results show that the proposed MU R-CNN is applicable to both high- and low-quality images, and thus more suitable for Industry 4.0.

The original version of this Article omitted an affiliation of Xiewen Wen: ‘College of Chemistry and Molecular Engineering, Beijing National Laboratory for Molecular Sciences, Peking University, Beijing 100871, China’. This has been corrected in both the PDF and HTML versions of the Article.

An off-line optimization approach of high precision minimum time feedrate for CNC machining is proposed. Besides the ordinary considered velocity, acceleration, and jerk constraints, dynamic performance constraint of each servo drive is also considered in this optimization problem to improve the tracking precision along the optimized feedrate trajectory. Tracking error is applied to indicate the servo dynamic performance of each axis. By using variable substitution, the tracking error constrained minimum time trajectory planning problem is formulated as a nonlinear path constrained optimal control problem. Bang-bang constraints structure of the optimal trajectory is proved in this paper; then a novel constraint handling method is proposed to realize a convex optimization based solution of the nonlinear constrained optimal control problem. A simple ellipse feedrate planning test is presented to demonstrate the effectiveness of the approach. Then the practicability and robustness of the trajectory generated by the proposed approach are demonstrated by a butterfly contour machining example.

Transcription factor E1AF is widely known to play critical roles in tumor metastasis via directly binding to the promoters of genes involved in tumor migration and invasion. Here, we report for the first time E1AF as a novel binding partner for ubiquitously expressed Sp1 transcription factor. E1AF forms a complex with Sp1, contributes to Sp1 phosphorylation and transcriptional activity, and functions as a mediator between epidermal growth factor and Sp1 phosphorylation and activity. Sp1 functions as a carrier bringing E1AF to the promoter region, thus activating transcription of glioma-related gene for ß1,4-galactosyltransferase V (GalT V; EC 2.4.1.38). Biologically, E1AF functions as a positive invasion regulator in glioma in cooperation with Sp1 partly via up-regulation of GalT V. This report describes a new mechanism of glioma invasion involving a cooperative effort between E1AF and Sp1 transcription factors. [ABSTRACT FROM AUTHOR]

Androgen receptor (AR) is essential for the maintenance of the male reproductive systems and is critical for the carcinogenesis of human prostate cancers (PCas). D-type cyclins are closely related to the repression of AR function. It has been well documented that cyclin D1 inhibits AR function through multiple mechanisms, but the mechanism of how cyclin D3 exerts its repressive role in the AR signaling pathway remains to be identified. In the present investigation, we demonstrate that cyclin D3 and the 58-kDa isoform of cyclin-dependent kinase 11 (CDK11p58) repressed AR transcriptional activity as measured by reporter assays of transformed cells and prostate-specific antigen expression in PCa cells. AR, cyclin D3, and CDK11p58 formed a ternary complex in cells and were colocalized in the luminal epithelial layer of the prostate. AR activity is controlled by phosphorylation at specific sites. We found that AR was phosphorylated at Ser-308 by cyclin D3/CDK11p58 in vitro and in vivo, leading to the repressed activity of AR transcriptional activation unit 1 (TAU1). Furthermore, androgen-dependent proliferation of PCa cells was inhibited by cyclin D3/CDK11p58 through AR repression. These data suggest that cyclin D3/CDK11p58 signaling is involved in the negative regulation of AR function. [ABSTRACT FROM AUTHOR]

The liver is the main site of estrogen metabolism, and liver disease is usually associated with an abnormal estrogen status. However, little is known about the mechanism underlying this connection. Here, we investigated the effects of bile acid (BA)- activated farnesoid X receptor (FXR) on the metabolism of 17β-estradiol (E2) during blockage of bile flow (cholestasis). Correlations between BA levels and E2 concentrations were established in patients with cholestasis, and hepatic expression profiles of key genes involved in estrogen metabolism were investigated in both WT and FXR-/- mice. We found that the elevated E2 level positively correlated withBAconcentrations in the patients with cholestasis.Wefurther observed that bile duct ligation (BDL) increases E2 levels in mouse serum, and this elevation effect was alleviated by deleting the FXR gene. Of note, FXR down-regulated the expression of hepatic sulfotransferase SULT1E1, the primary enzyme responsible for metabolic estrogen inactivation. At the molecular level, we found that FXR competes with the protein acetylase CREB-binding protein (CBP) for binding to the transcription factor hepatocyte nuclear factor 4α (HNF4α). This competition decreased HNF4α acetylation and nuclear retention, which, in turn, repressed HNF4α-dependent SULT1E1 gene transcription. These findings suggest that cholestasis induces BA-activated FXR activity, leading to downstream inhibition of SULT1E1 and hence impeding hepatic degradation of estrogen. [ABSTRACT FROM AUTHOR]

Deregulated expression of N-acetylgalactosaminyltransferases (GALNTs), which is responsible for the initial step of mucin-type O-glycosylation, could produce abnormal truncated O-glycans and thereby exert pivotal functions during malignant transformation. GALNT4 is one of the few isoforms preferring to catalyze partial GalNAc-glycosylated substrates and modify the sites not utilized by other known GALNTs. This study aims to evaluate the impact of GALNT4 expression on malignant transformation of hepatocellular carcinoma (HCC). Immunohistochemistry and in situ hybridization analysis were performed to assess GALNT4 and miR-9 level in clinical specimens, respectively. GALNT4 expression is markedly repressed in primary HCC tissues, and reduced expression of GALNT4 is significantly associated with adverse survival of patients with HCC. Functional investigations demonstrate that repressed GALNT4 could promote migration, invasion, anoikis resistance, and stemness of HCC cells in vitro as well as tumor growth in vivo. The wild-type GALNT4 could modify O-linked glycosylation on EGFR and thus modulate the activity of EGFR. A luciferase activity assay further identified microRNA-9 (miR-9) as the crucial specific arbitrator for GALNT4 expression in HCC cells. Furthermore, restoring GALNT4 expression attenuates miR-9-mediated oncogenic functions. Kaplan-Meier survival analysis indicates that the miR-9/GALNT4 expression signature yields promising prognostic significance to refine the risk stratification of patients with HCC. In conclusion, this study establishes the miR-9/GALNT4 axis as a potential adverse prognostic factor and therapeutic target for HCC patients. [ABSTRACT FROM AUTHOR]

Gastric cancer remains the third leading cause of cancerrelated mortality worldwide, and invasion and metastasis of gastric cancer represent the major reason for its poor prognosis. In this study, we found that loss of the receptor for activated C-kinase 1 (RACK1) promoted the metastasis of gastric cancer by enhancing the autocrine of IL8 in vitro and in vivo. microRNA (miRNA; miR) array identified that RACK1 modulated the expression of a series of miRNAs, including the miR-302 cluster, and RACK1 modulated the IL8 expression and tumor invasion through miRNA-302c. Moreover, upregulation of IL8 in turn decreased the level of miRNA-302c and induced IL8 expression in a feedback manner. Tissue microarray also indicated that RACK1 was correlated with invasion/metastasis phenotype, IL8 expression, as well as 5-year survival in clinical cases of gastric cancer. Together, our results imply that loss of RACK1 in gastric cancer links epigenetics to inflammatory cytokines to promote tumor metastasis. [ABSTRACT FROM AUTHOR]

Fructose is a key dietary factor in the development of nonalcoholic fatty liver disease (NAFLD). Here we investigated whether WAY-362450 (WAY), a potent synthetic and orally active FXR agonist, protects against fructose-induced steatosis and the underlying mechanisms. C57BL/6J mice, fed 30% fructose for 8 weeks, were treated with or without WAY, 30 mg/kg, for 20 days. The elevation of serum and hepatic triglyceride in mice fed 30% fructose was reversed by WAY treatment. Histologically, WAY significantly reduced triglyceride accumulation in liver, attenuated microphage infiltration and protected the junction integrity in intestine. Moreover, WAY remarkably decreased portal endotoxin level, and lowered serum TNFα concentration. In lipopolysaccharide (LPS)-induced NAFLD model, WAY attenuated serum TNFα level. Moreover, WAY suppressed LPS-induced expression of hepatic lipid droplet protein adipose differentiation-related protein (ADRP), down-regulation of it in mice fed 30% fructose. Furthermore, WAY repressed lipid accumulation and ADRP expression in a dose-dependent manner in palmitic acid (PA)-treated HepG2 and Huh7 cells. WAY suppressed TNFα-induced ADRP up-regulation via competing with AP-1 for ADRP promoter binding region. Together, our findings suggest that WAY, an FXR agonist, attenuates liver steatosis through multiple mechanisms critically involved in the development of hepatosteatosis, and represents a candidate for NAFLD treatment. [ABSTRACT FROM AUTHOR]

Coordinated translation initiation is coupled with cell cycle progression and cell growth, whereas excessive ribosome biogenesis and translation initiation often lead to tumor transformation and survival. Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide and generally displays inherently high resistance to chemotherapeutic drugs. We found that RACK1, the receptor for activated C-kinase 1, was highly expressed in normal liver and frequently upregulated in HCC. Aberrant expression of RACK1 contributed to in vitro chemoresistance as well as in vivo tumor growth of HCC. These effects depended on ribosome localization of RACK1. Ribosomal RACK1 coupled with PKCβII to promote the phosphorylation of eukaryotic initiation factor 4E (eIF4E), which led to preferential translation of the potent factors involved in growth and survival. Inhibition of PKCβII or depletion of eIF4E abolished RACK1-mediated chemotherapy resistance of HCC in vitro. Our results imply that RACK1 may function as an internal factor involved in the growth and survival of HCC and suggest that targeting RACK1 may be an efficacious strategy for HCC treatment. [ABSTRACT FROM AUTHOR]

c-Src activates Ras-MAPK/ERK signaling pathway and regulates cell migration, while trihydrophobin 1 (TH1) inhibits MAPK/ ERK activation and cell migration through interaction with A-Raf and PAK1 and inhibiting their kinase activities. Here we show that c-Src interacts with TH1 by GST-pull down assay, coimmunoprecipitation and confocal microscopy assay. The interaction leads to phosphorylation of TH1 at Tyr-6 in vivo and in vitro. Phosphorylation of TH1 decreases its association with A-Raf and PAK1. Further study reveals that Tyr-6 phosphorylation of TH1 reduces its inhibition on MAPK/ERK signaling, enhances c-Src mediated cell migration. Moreover, induced tyrosine phosphorylation of TH1 has been found by EGF and estrogen treatments. Taken together, our findings demonstrate a novel mechanism for the comprehensive regulation of Ras/Raf/MEK/ERK signaling and cell migration involving tyrosine phosphorylation of TH1 by c-Src. [ABSTRACT FROM AUTHOR]

Of the three envelope glycoproteins encoded by hepatitis B virus (HBV) that are collectively referred to as HBV surface antigen (HBsAg), the large HBsAg (LHBs) glycoprotein is expressed preferentially in HBV-associated hepatocellular carcinoma. LHBs can act as an oncogene in transgenic mice, but how it contributes functionally to hepatocarcinogenesis remains unclear. In this study, we determined the molecular and functional roles of LHBs during HBV-associated hepatocarcinogenesis. LHBs increased tumor formation of hepatoma cells. Moreover, expression of LHBs but not other HBV envelope glycoproteins specifically promoted proliferation of hepatoma and hepatic cells in vitro. Mechanistic investigations revealed that these effects were caused by activation of the Src/PI3K/Akt pathway through proximal stimulation of PKCα/Raf1 signaling by LHBs. Proliferation induced by stable LHBs expression was associated with increased G1-S cell-cycle progression and apoptosis resistance mediated by Src kinase activation, as established in hepatocellular carcinoma clinical specimens. Importantly, LHBs-induced cellular proliferation and tumor formation were reversed by administration of the Src inhibitor saracatinib. Together, our findings suggest that LHBs promotes tumorigenesis of hepatoma cells by triggering a PKCα/Raf1 to Src/PI3K/Akt signaling pathway, revealing novel insights into the underlying mechanisms of HBV-associated hepatocarcinogenesis. [ABSTRACT FROM AUTHOR]

CDK11p58, a member of the p34cdc2-related kinase family, is associated with cell cycle progression, tumorigenesis, and proapoptotic signaling. It is also required for the maintenance of chromosome cohesion, the maturation of centrosome, the formation of bipolar spindle, and the completion of mitosis. Here we identified that CDK11p58 interacted with itself to form homodimers in cells, whereas D224N, the kinase-dead mutant, failed to form homodimers. CDK11p58 was autophosphorylated, and the main functions of CDK11p58, such as kinase activity, transactivation of nuclear receptors, and proapoptotic signal transduction, were dependent on its autophosphorylation. Furthermore, the in vitro kinase assay indicated that CDK11p58 was autophosphorylated at Thr-370. By mutagenesis, we created CDK11p58 T370A and CDK11p58 T370D, which mimic the dephosphorylated and phosphorylated forms of CDK11p58, respectively. The T370A mutant could not form dimers and be phosphorylated by the wild type CDK11p58 and finally lost the kinase activity. Further functional research revealed that T370A failed to repress the transactivition of androgen receptor and enhance the cell apoptosis. Overall, our data indicated that Thr-370 is responsible for the autophosphorylation, dimerization, and kinase activity of CDK11p58. Moreover, Thr-370 mutants might affect CDK11p58-mediated signaling pathways. [ABSTRACT FROM AUTHOR]

ATFS, a member of activating transcription factor (ATF)/ cAMP-response element-binding protein (CREB) family of b-ZIP transcription factors, contributes to neural cell differentiation and is involved in cell apoptosis in response to cisplatin and a number of environment factors. However, the mechanisms governing the regulation of ATF5 protein during apoptosis are largely unknown. In this study we reported that ATF5 protein was a substrate of the ubiquitin-proteasome pathway. Interestingly, the ubiqttitin-dependent degradation of exogenous ATF5 protein was independent of lysine residues. Instead, the addition of a large N-terminal enhanced green fluorescence protein tag increased the stability of ATF5 protein, and the free amino acid group of the N-terminal methionine of ATF5 protein was a site for ubiquitinylation, indicating that exogenous ATF5 was degraded via the ubiquitin-proteasome system through N-terminal ubiquitinylation. Furthermore, cisplatin increased ATF5 protein expression via preventing its ubiquitin-dependent degradation, which might be associated with its promoting the nucleus-to-cytoplasm translocation of E2 ubiqnitin-conjugating enzyme Cdc34 and reducing the interaction between ATF5 and Cdc34. In summary, a down-regulation of proteasome-mediated degradation of ATF5 might contribute to cisplatin-indnced apoptosis, providing a new mechanism of cisplatin-induced apoptosis. [ABSTRACT FROM AUTHOR]

β1,4-gaiactosyltransferase V (GAlTV; EC 2.4.1.38) can effectively galactosylate the GlcNAcβ1→6Man arm of the highly branched N-glycans that are characteristic of glioma. Previously, we have reported that the expression of GalT V is increased in the process of glioma. However, currently little is known about the role of GAlT V in this process. In this study, the ectopic expression of GalT V could promote the invasion and survival of glioma cells and transformed astrocytes. Furthermore, decreasing the expression of GalT V in glioma cells promoted apoptosis, inhibited the Invasion and migration and the ability of tumor formation in vivo, and reduced the activation of AKT. In addition, the activity of GalT V promoter could be induced by epidermal growth factor, dominant active Ras, ERK1, JNK1, and constitutively active AKT. Taken together, our results suggest that GalT V functioned as a novel glioma growth activator and might represent a novel target in glioma therapy. [ABSTRACT FROM AUTHOR]

The elevated levels of β1,4.galactosyltransferase I (GalT I; EC 2.4.1.38) are detected in highly metastatic lung cancer PGBE1 cells compared with its less metastatic partner PGLH7 cells. Decreasing the GalT I surface expression by small interfering RNA or interfering with the surface of GalT I function by mutation inhibited cell adhesion on laminin, the invasive potential in vitro, and tyrosine phosphorylation of focal adhesion kinase. The mechanism by which GalT I activity is up-regulated in highly metastatic cells remains unclear. To investigate the regulation of Galt I expression, we cloned the 5′-region flanking the transcription start point of the GalT I gene (-1653 to +52). Cotransfection of the GalT I promoter/luciferase reporter and the Ets family protein E1AF expression plasmid increased the luciferase reporter activity in a dose-dependent manner. By deletion and mutation analyses, we identified an Ets-binding site between nucleotides -205 and -200 in the GalT I promoter that was critical for responsiveness to E1AF. It was identified that E1AF could bind to and activate the GalT I promoter by electrophoretic mobility shift assay in PGLH7 cells and COS1 cells. A stronger affirnity of E1AF for DNA has contributed to the elevated expression of GalT I in PGBE1 cells. Stable transfection of the E1AF expression plasmid resulted in increased GalT I expression in PGLH7 cells, and stable transfectants migrated faster than control cells. Meanwhile, the content of the β1,4-Gal branch on the cell surface was increased in stably transfected PGLH7 cells. GalT I expression can also be induced by epidermal growth factor and dominant active Ras, JNK1, and ERK1. These data suggest an essential role for E1AF in the activation of the human GalT I gene in highly metastatic lung cancer cells. [ABSTRACT FROM AUTHOR]

Our previous work indicated that instead of binding to B-Raf or C-Raf, trihydrophobin 1 (TH1) specifically binds to A-Raf kinase both in vitro and in vivo. In this work, we investigated its function further. Using confocal microscopy, we found that TH1 colocalizes with A-Raf, which confirms our former results. The region of TH1 responsible for the interaction with A-Raf is mapped to amino acids 1-372. Coimmunoprecipitation experiments demonstrate that TH1 is associated with A-Raf in both quiescent and serum-stimulated cells. Wild type A-Raf binds increasingly to TH1 when it is activated by serum and/or upstream oncogenic Ras/Src compared with that of "kinase-dead" A-Raf. The latter can still bind to TH1 under the same experimental condition. The binding pattern of A-Raf implies that this interaction is mediated in part by the A-Raf kinase activity. As indicated by Raf protein kinase assays, TH1 inhibits A-Raf kinase, whereas neither B-Raf nor C-Raf kinase activity is influenced. Furthermore, we observed that TH1 inhibited cell cycle progression in TH1 stably transfected 7721 cells compared with mock cells, and flow cell cytometry analysis suggested that the TH1 stably transfected 7721 cells were G0/G1 phase-arrested. Taken together, our data provide a clue to understanding the cellular function of TH1 on Raf isoform-specific regulation. [ABSTRACT FROM AUTHOR]

Investigates the molecular mechanism of apoptosis mediated by PITSLRE protein p110C. Transient transfection; In vitro binding analysis and interaction assay; Fluorescence imaging of living cells.