Analysis of polarized secretion of fucosylated alpha-fetoprotein in HepG2 cells

Fucosylated alpha-fetoprotein (AFP) is a more specific biomarker for hepatocellular carcinoma (HCC) than AFP. However, the mechanisms underlying the increase in fucosylated AFP in sera of HCC patients remain largely unknown. Recently, we reported that fucosylation is a possible signal for the secretion of hepatic glycoproteins into bile and that the fucosylation-based sorting machinery might be disrupted in the liver bearing HCC. In this study, we investigated the selective secretion of fucosylated AFP into bile canaliculus (BC) structures of the human hepatoma cell line HepG2. The proportion of fucosylated AFP in BC structures was higher than that in the medium, as judged by lectin affinity electrophoresis. Suppression of fucosylation by the double knock-down of GDP-mannose-4,6-dehydratase and the human homologue of GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase, which contribute to the synthesis of GDP-fucose, a donor substrate for fucosyltransferases, did not decrease the proportion of fucosylated AFP in BC structures but decreased this proportion in conditioned medium. Furthermore, increased AFP fucosylation was observed in medium, but not in BC structures, upon adding free fucose. These results suggest that saturation of fucosylated AFP in BC structures is accompanied by its increase in conditioned medium, probably leading to increased fucosylated AFP in sera of HCC patients.     

Glycosidic inhibitors of melanogenesis from leaves of Momordica charantia

Eight glycosidic compounds, 1-8, including two new compounds, (4ξ)-α-terpineol 8-O-[α-L-arabinopyranosyl-(1→6)-β-D-glucopyranoside] (5) and myrtenol 10-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside] (7), were isolated from the BuOH-soluble fraction of a MeOH extract of Momordica charantia leaves. The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses and comparison with literature. Upon evaluation of compounds 1-8 on the melanogenesis in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), these compounds were found to exhibit inhibitory activities with 7.1-27.0% and 23.6-46.4% reduction of melanin content at 30 μM and 100 μM, respectively, with no or almost no toxicity to the cells (80.0-103.5% of cell viability at 100 μM). Western blot analysis showed that compound 7 reduced the protein levels of MITF, tyrosinase, TRP-1, and TRP-2 mostly in a concentration-dependent manner, suggesting that this compound inhibits melanogenesis on the α-MSH-stimulated B16 melanoma cells by, at least in part, inhibiting the expression of MITF, followed by decreasing the expression of tyrosinase, TRP-1, and TRP-2.     

Altered Plasma Apolipoprotein Modifications in Patients with Pancreatic Cancer: Protein Characterization and Multi-Institutional Validation

Background

Among the more common human malignancies, invasive ductal carcinoma of the pancreas has the worst prognosis. The poor outcome seems to be attributable to difficulty in early detection.

Methods

We compared the plasma protein profiles of 112 pancreatic cancer patients with those of 103 sex- and age-matched healthy controls (Cohort 1) using a newly developed matrix-assisted laser desorption/ionization (oMALDI) QqTOF (quadrupole time-of-flight) mass spectrometry (MS) system.

Results

We found that hemi-truncated apolipoprotein AII dimer (ApoAII-2; 17252 m/z), unglycosylated apolipoprotein CIII (ApoCIII-0; 8766 m/z), and their summed value were significantly decreased in the pancreatic cancer patients [P = 1.36×10⁻²¹, P = 4.35×10⁻¹⁴, and P = 1.83×10⁻²⁴ (Mann-Whitney U-test); area-under-curve values of 0.877, 0.798, and 0.903, respectively]. The significance was further validated in a total of 1099 plasma/serum samples, consisting of 2 retrospective cohorts [Cohort 2 (n = 103) and Cohort 3 (n = 163)] and a prospective cohort [Cohort 4 (n = 833)] collected from 8 medical institutions in Japan and Germany.

Conclusions

We have constructed a robust quantitative MS profiling system and used it to validate alterations of modified apolipoproteins in multiple cohorts of patients with pancreatic cancer.     

Up-Regulation of Imp3 Confers In Vivo Tumorigenicity on Murine Osteosarcoma Cells

Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.     

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity.     

N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.     

Cardiac mTOR protects the heart against ischemia-reperfusion injury

Cardiac mammalian target of rapamycin (mTOR) is necessary and sufficient to prevent cardiac dysfunction in pathological hypertrophy. However, the role of cardiac mTOR in heart failure after ischemic injury remains undefined. To address this question, we used transgenic (Tg) mice with cardiac-specific overexpression of mTOR (mTOR-Tg mice) to study ischemia-reperfusion (I/R) injury in two animal models: 1) in vivo I/R injury with transient coronary artery ligation and 2) ex vivo I/R injury in Langendorff-perfused hearts with transient global ischemia. At 28 days after I/R, mortality was lower in mTOR-Tg mice than littermate control mice [wild-type (WT) mice]. Echocardiography and MRI demonstrated that global cardiac function in mTOR-Tg mice was preserved, whereas WT mice exhibited significant cardiac dysfunction. Masson's trichrome staining showed that 28 days after I/R, the area of interstitial fibrosis was smaller in mTOR-Tg mice compared with WT mice, suggesting that adverse left ventricular remodeling is inhibited in mTOR-Tg mice. In the ex vivo I/R model, mTOR-Tg hearts demonstrated improved functional recovery compared with WT hearts. Perfusion with Evans blue after ex vivo I/R yielded less staining in mTOR-Tg hearts than WT hearts, indicating that mTOR overexpression inhibited necrosis during I/R injury. Expression of proinflammatory cytokines, including IL-6 and TNF-α, in mTOR-Tg hearts was lower than in WT hearts. Consistent with this, IL-6 in the effluent post-I/R injury was lower in mTOR-Tg hearts than in WT hearts. These findings suggest that cardiac mTOR overexpression in the heart is sufficient to provide substantial cardioprotection against I/R injury and suppress the inflammatory response.     

Kinase-dead ATM protein causes genomic instability and early embryonic lethality in mice

Studies on cell division traditionally focus on the mechanisms of chromosome segregation and cytokinesis, yet we know comparatively little about how organelles segregate. Analysis of organelle partitioning in asymmetrically dividing cells has provided insights into the mechanisms through which cells control organelle distribution. Interestingly, these studies have revealed that segregation mechanisms frequently link organelle distribution to organelle growth and formation. Furthermore, in many cases, cells use organelles, such as the endoplasmic reticulum and P granules, as vectors for the segregation of information. Together, these emerging data suggest that the coordination between organelle growth, division, and segregation plays an important role in the control of cell fate inheritance, cellular aging, and rejuvenation, i.e., the resetting of age in immortal lineages.     

Elevated expression of brain‐derived neurotrophic factor facilitates visual imprinting in chicks

With the aim of elucidating the neural mechanisms of early learning, we studied the role of brain‐derived neurotrophic factor (BDNF) in visual imprinting in birds. The telencephalic neural circuit connecting the visual Wulst and intermediate medial mesopallium is critical for imprinting, and the core region of the hyperpallium densocellulare (HDCo), situated at the center of this circuit, has a key role in regulating the activity of the circuit. We found that the number of BDNF mRNA‐positive cells in the HDCo was elevated during the critical period, particularly at its onset, on the day of hatching (P0). After imprinting training on P1, BDNF mRNA‐positive cells in the HDCo increased in number, and tyrosine phosphorylation of TrkB was observed. BDNF infusion into the HDCo at P1 induced imprinting, even with a weak training protocol that does not normally induce imprinting. In contrast, K252a, an antagonist of Trk, inhibited imprinting. Injection of BDNF at P7, after the critical period, did not elicit imprinting. These results suggest that BDNF promotes the induction of imprinting through TrkB exclusively during the critical period.