A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca(2+)-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 μM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca(2+)](i)-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca(2+)](i) increases.     

STT3B-dependent posttranslational N-glycosylation as a surveillance system for secretory protein

The chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways-MMS2-dependent template switch and Pol ζ-dependent bypass-are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1-4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.     

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3'), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.     

Diffuse large B-cell lymphoma presenting with neurolymphomatosis and intravascular lymphoma: a unique autopsy case with diverse neurological symptoms

ABSTRACT: A 78-year-old Japanese male noticed a difficulty in the beginning of standing up, followed by a progressive numbness of extremities with pain, Bell's palsy, dysarthria, and difficulty in swallowing. A clinician had suspected cancer of unknown primary origin, accompanied by the diverse and elusive neurological symptoms, likely presenting as painful mononeuropathy simplex and cranial neuropathy. He developed dysbasia over weeks and died 1 month after the symptom onset. At autopsy, an ill-defined large and soft tumor mass in the right lobe of the liver with direct invasion into the right adrenal gland was observed. The left adrenal gland or right iliopsoas muscle was also involved. Microscopic findings showed a monotonous proliferation of medium-sized to large atypical lymphoid cells, which were diffusely positive for CD20 in immunohistochemistry, consistent with diffuse large B-cell lymphoma (DLBL). Furthermore, the lymphoma cells aggressively infiltrated endoneurial and subperineurial spaces not only in the peripheral nerves and plexuses, but partly in the spinal nerve roots, and intravascular spaces in various tissues. Therefore, systemic lymphoma (DLBL) complicated with neurolymphomatosis (NL) and intravascular lymphoma (IVL) was diagnosed. Very early diagnosis and treatment are necessary for the NL patients with poor prognosis. Virtual slides The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5862472377020448.     

Identification, characterization, and developmental regulation of two storage proteins in the bamboo borer Omphisa fuscidentalis

Two insect storage proteins, OfSP1 (75 kDa) and OfSP2 (72 kDa), were purified using three different chromatographies from the hemolymph of Omphisa fuscidentalis larvae during diapause, and their genes were cloned. OfSP1 and OfSP2 concentrations in the hemolymph were high during diapause. During pupation, OfSP1 levels decreased in the male hemolymph and disappeared from the female hemolymph. OfSP1 and OfSP2 mRNA levels in the fat bodies were low during the third instar, but increased greatly during the fourth and fifth larval instars. During diapause, mRNA expression continued at a lower level than during the feeding period. The injection of 20-hydroxyecdysone (20E) into diapausing larvae caused an increase in OfSP1 and OfSP2 mRNA levels 2-3 days post-injection, followed by a decrease in expression until pupation, which occurred 2-4 days thereafter. When larvae were treated with juvenile-hormone analog (JHA), OfSP1 and OfSP2 mRNA levels gradually decreased until the onset of pupation. In Omphisa, OfSP1 and OfSP2 proteins are produced and released by the larval fat bodies in the fourth and fifth-instar larvae, and the proteins accumulate in the hemolymph until the insects enter diapause. OfSP1 may be reabsorbed by the fat bodies at the end of diapause for subsequent re-use during pupation.     

YIPF5 and YIF1A recycle between the ER and the Golgi apparatus and are involved in the maintenance of the Golgi structure

Yip1p/Yif1p family proteins are five-span transmembrane proteins localized in the Golgi apparatus and the ER. There are nine family members in humans, and YIPF5 and YIF1A are the human orthologs of budding yeast Yip1p and Yif1p, respectively. We raised antisera against YIPF5 and YIF1A and examined the localization of endogenous proteins in HeLa cells. Immunofluorescence, immunoelectron microscopy and subcellular fractionation analysis suggested that YIPF5 and YIF1A are not restricted to ER exit sites but also localized in the ER-Golgi intermediate compartment (ERGIC) and some in the cis-Golgi at steady state. Along with ERGIC53, YIPF5 and YIF1A remained in the cytoplasmic punctate structures after brefeldin A treatment, accumulated in the ERGIC and the cis-Golgi after treatment with AlF₄^- and accumulated in the ER when ER to Golgi transport was inhibited by Sar1(H79G). These results supported the localization of YIPF5 and YIF1A in the ERGIC and the cis-Golgi, and strongly suggested that they are recycling between the ER and the Golgi apparatus. Analysis by blue native PAGE and co-immunoprecipitation showed that YIPF5 and YIF1A form stable complexes of three different sizes. Interestingly, the knockdown of YIPF5 or YIF1A caused partial disassembly of the Golgi apparatus suggesting that YIPF5 and YIF1A are involved in the maintenance of the Golgi structure.     

Hyperosmotic stress up-regulates the expression of major vault protein in SW620 human colon cancer cells

The major vault protein (MVP) is the major constituent of the vault particle, the largest ribonuclear protein complex described to date and is identical to lung resistance-related protein (LRP). Although MVP is also expressed in several normal tissues, little is known about its physiological role. MVP played a protective role against some xenobiotics and other stresses. We thus investigated the effect of osmotic stress on MVP expression by treating human colon cancer SW620 cells with sucrose or NaCl. The expression level of both MVP protein and MVP mRNA was increased by the osmostress. Sucrose or sodium chloride could also enhance MVP promoter activity. Inhibition of p38 MAPK in SW620 cells by SB203580 inhibited the expression of MVP under hyperosmotic stress. These findings suggested that osmotic stress up-regulated the MVP expression through p38 MAPK pathway. Down-regulation of MVP expression by MVP interfering RNA (RNAi) in SW620 cells increased the sensitivity of the cells to hyperosmotic stress and enhanced apoptosis. Furthermore, MVP RNAi prevented the osmotic stress-induced, time-dependent increase in phosphorylated Akt. These findings suggest that the PI3K/Akt pathway might be implicated in the cytoprotective effect of MVP.Our data demonstrate that exposure of cells to hyperosmotic stress induces MVP that might play an important role in the protection of the cells from the adverse effects of osmotic stress.