Aeromonas hydrophila exotoxin induces cytoplasmic vacuolation and cell death in VERO cells.

PubMed

Di Pietro, Angela; Picerno, Isa; Visalli, Giuseppa; Chirico, Cristina; Spataro, Pasquale; Cannavò, Giuseppe; Scoglio, Maria E

2005-07-01

Many organisms are able to cause cell vacuolation, but it is unclear if this can be considered a step of apoptosis or necrosis, or a distinct form of cell death. In this study VERO cells were used to evaluate the relationship between vacuolation and cell death pattern caused by exotoxins produced by environmental strains of A. hydrophila. Cell damage has been evaluated morphologically as well as biochemically. Cytotoxic and vacuolating titres were strictly correlated and the vacuolation has to be considered an early indicator of cytotoxicity that causes cell apoptosis or necrosis in relation to the dose. Signs of apoptosis (chromatin condensation and blebbing) were observed at low concentration and TGase activity, referable to apoptosis induction, confirms morphological observations. In fact, putrescine incorporation was related both to cytotoxin concentration and time of incubation. Moreover, the observed doubling cells with necrotic features permit us to suppose that cell sensitivity and death pattern could change during the different phases of cellular cycle.

[Pancreatic acinar neoplasms : Comparative molecular characterization].

PubMed

Bergmann, F

2016-11-01

Pancreatic acinar cell carcinomas are biologically aggressive neoplasms for which treatment options are very limited. The molecular mechanisms of tumor initiation and progression are largely not understood and precursor lesions have not yet been identified. In this study, pancreatic acinar cell carcinomas were cytogenetically characterized as well as by molecular and immunohistochemical analyses. Corresponding investigations were carried out on pancreatic ductal adenocarcinomas and pancreatic neuroendocrine neoplasms augmented by functional analyses. We show that pancreatic acinar cell carcinomas display a microsatellite stable, chromosomal unstable genotype, characterized by recurrent chromosomal imbalances that clearly discriminate them from pancreatic ductal adenocarcinomas and neuroendocrine neoplasms. Based on findings obtained from comparative genomic hybridization, candidate genes could be identified, such as deleted in colorectal cancer (DCC) and c-MYC. Furthermore, several therapeutic targets were identified in acinar cell carcinomas and other pancreatic neoplasms, including epidermal growth factor receptor (EGFR), L1 cell adhesion molecule (L1CAM) and heat shock protein 90 (HSP90). Moreover, L1CAM was shown to play a significant role in the tumorigenesis of pancreatic ductal adenocarcinoma. Functional analyses in cell lines derived from pancreatic neuroendocrine neoplasms revealed promising anti-tumorigenic effects using EGFR and HSP90 inhibitors affecting the cell cycle and in the case of HSP90, regulating several other oncogenes. Finally, based on mutational analyses of mitochondrial DNA, molecular evidence is provided that acinar cell cystadenomas (or better cystic acinar transformation) represent non-clonal lesions, suggesting an inflammatory reactive non-neoplastic nature.

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.

PubMed

Muili, Kamaldeen A; Ahmad, Mahwish; Orabi, Abrahim I; Mahmood, Syeda M; Shah, Ahsan U; Molkentin, Jeffery D; Husain, Sohail Z

2012-04-15

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury

PubMed Central

Muili, Kamaldeen A.; Ahmad, Mahwish; Orabi, Abrahim I.; Mahmood, Syeda M.; Shah, Ahsan U.; Molkentin, Jeffery D.

2012-01-01

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca2+ is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca2+ signaling is the Ca2+-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ−/−) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis. PMID:22323127

Induced PTF1a expression in pancreatic ductal adenocarcinoma cells activates acinar gene networks, reduces tumorigenic properties, and sensitizes cells to gemcitabine treatment.

PubMed

Jakubison, Brad L; Schweickert, Patrick G; Moser, Sarah E; Yang, Yi; Gao, Hongyu; Scully, Kathleen; Itkin-Ansari, Pamela; Liu, Yunlong; Konieczny, Stephen F

2018-05-02

Pancreatic acinar cells synthesize, package, and secrete digestive enzymes into the duodenum to aid in nutrient absorption and meet metabolic demands. When exposed to cellular stresses and insults, acinar cells undergo a dedifferentiation process termed acinar-ductal metaplasia (ADM). ADM lesions with oncogenic mutations eventually give rise to pancreatic ductal adenocarcinoma (PDAC). In healthy pancreata, the basic helix-loop-helix (bHLH) factors MIST1 and PTF1a coordinate an acinar-specific transcription network that maintains the highly developed differentiation status of the cells, protecting the pancreas from undergoing a transformative process. However, when MIST1 and PTF1a gene expression is silenced, cells are more prone to progress to PDAC. In this study, we tested whether induced MIST1 or PTF1a expression in PDAC cells could (i) re-establish the transcriptional program of differentiated acinar cells and (ii) simultaneously reduce tumor cell properties. As predicted, PTF1a induced gene expression of digestive enzymes and acinar-specific transcription factors, while MIST1 induced gene expression of vesicle trafficking molecules as well as activation of unfolded protein response components, all of which are essential to handle the high protein production load that is characteristic of acinar cells. Importantly, induction of PTF1a in PDAC also influenced cancer-associated properties, leading to a decrease in cell proliferation, cancer stem cell numbers, and repression of key ATP-binding cassette efflux transporters resulting in heightened sensitivity to gemcitabine. Thus, activation of pancreatic bHLH transcription factors rescues the acinar gene program and decreases tumorigenic properties in pancreatic cancer cells, offering unique opportunities to develop novel therapeutic intervention strategies for this deadly disease. © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Salivary gland acinar cells regenerate functional glandular structures in modified hydrogels

NASA Astrophysics Data System (ADS)

Pradhan, Swati

Xerostomia, a condition resulting from irradiation of the head and neck, affects over 40,000 cancer patients each year in the United States. Direct radiation damage of the acinar cells that secrete fluid and protein results in salivary gland hypofunction. Present medical management for xerostomia for patients treated for upper respiratory cancer is largely ineffective. Patients who have survived their terminal diagnosis are often left with a diminished quality of life and are unable to enjoy the simple pleasures of eating and drinking. This project aims to ultimately reduce human suffering by developing a functional implantable artificial salivary gland. The goal was to create an extracellular matrix (ECM) modified hyaluronic acid (HA) based hydrogel culture system that allows for the growth and differentiation of salivary acinar cells into functional acini-like structures capable of secreting large amounts of protein and fluid unidirectionally and to ultimately engineer a functional artificial salivary gland that can be implanted into an animal model. A tissue collection protocol was established and salivary gland tissue was obtained from patients undergoing head and neck surgery. The tissue specimen was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Hematoxylin and eosin staining confirmed normal glandular tissue structures including intercalated ducts, striated ducts and acini. alpha-Amylase and periodic acid schiff stain, used for structures with a high proportion of carbohydrate macromolecules, preferentially stained acinar cells in the tissue. Intercalated and striated duct structures were identified using cytokeratins 19 and 7 staining. Myoepithelial cells positive for cytokeratin 14 were found wrapped around the serous and mucous acini. Tight junction components including ZO-1 and E-cadherin were present between both ductal and acinar cells. Ductal and acinar

Polycystin-2 Expression and Function in Adult Mouse Lacrimal Acinar Cells

PubMed Central

Hilgenberg, Jill D.; Rybalchenko, Volodymyr; Medina-Ortiz, Wanda E.; Gregg, Elaine V.; Koulen, Peter

2011-01-01

Purpose. Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca2+ homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca2+ concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP3Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca2+ release channels, transient receptor potential polycystin family (TRPP) channels. Methods. Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca2+ channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system. Results. All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues. Conclusions. The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology. PMID:21508103

Live morphological analysis of taxol-induced cytoplasmic vacuolization [corrected] in human lung adenocarcinoma cells.

PubMed

Wang, Xiao-Ping; Chen, Tong-Sheng; Sun, Lei; Cai, Ji-Ye; Wu, Ming-Qian; Mok, Martin

2008-12-01

Taxol (paclitaxel), one of the most active cancer chemotherapeutic agents, can cause programmed cell death (PCD) and cytoplasmic vacuolization. The objective of this study was to analyze the morphological characteristics induced by taxol. Human lung adenocarcinoma (ASTC-a-1) cells were exposed to various concentration of taxol. CCK-8 was used to assay the cell viability. Atomic force microscopy (AFM), plasmid transfection and confocal fluorescence microscopy were performed to image the cells morphological change induced by taxol. Fluorescence resonance energy transfer (FRET) was used to monitor the caspase-3 activation in living cells during taxol-induced cell death. Cells treated with taxol exhibited significant swelling and cytoplasmic vacuolization which may be due to endoplasmic reticulum (ER) vacuolization. Caspase-3 was not activated during taxol-induced cytoplasmic vacuolization and cell death. These findings suggest that taxol induces caspase-3-independent cytoplasmic vacuolization, cell swelling and cell death through ER vacuolization.

The relationship between vacuolation and initiation of PCD in rice (Oryza sativa) aleurone cells

NASA Astrophysics Data System (ADS)

Zheng, Yan; Zhang, Heting; Deng, Xiaojiang; Liu, Jing; Chen, Huiping

2017-01-01

Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.

Expression and subcellular localization of the ryanodine receptor in rat pancreatic acinar cells.

PubMed Central

Leite, M F; Dranoff, J A; Gao, L; Nathanson, M H

1999-01-01

The ryanodine receptor (RyR) is the principal Ca2+-release channel in excitable cells, whereas the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) is primarily responsible for Ca2+ release in non-excitable cells, including epithelia. RyR also is expressed in a number of non-excitable cell types, but is thought to serve as an auxiliary or alternative Ca2+-release pathway in those cells. Here we use reverse transcription PCR to show that a polarized epithelium, the pancreatic acinar cell, expresses the type 2, but not the type 1 or 3, isoform of RyR. We furthermore use immunochemistry to demonstrate that the type 2 RyR is distributed throughout the basolateral and, to a lesser extent, the apical region of the acinar cell, but is excluded from the trigger zone, where cytosolic Ca2+ signals originate in this cell type. Since propagation of Ca2+ waves in acinar cells is sensitive to ryanodine, caffeine and Ca2+, these findings suggest that Ca2+ waves in this cell type result from the co-ordinated release of Ca2+, first from InsP3Rs in the trigger zone, then from RyRs elsewhere in the cell. RyR may play a fundamental role in Ca2+ signalling in polarized epithelia, including for Ca2+ signals initiated by InsP3. PMID:9882629

Up-regulation of Store-operated Ca2+ Entry and Nuclear Factor of Activated T Cells Promote the Acinar Phenotype of the Primary Human Salivary Gland Cells*

PubMed Central

Jang, Shyh-Ing; Ong, Hwei Ling; Liu, Xibao; Alevizos, Ilias; Ambudkar, Indu S.

2016-01-01

The signaling pathways involved in the generation and maintenance of exocrine gland acinar cells have not yet been established. Primary human salivary gland epithelial cells, derived from salivary gland biopsies, acquired an acinar-like phenotype when the [Ca2+] in the serum-free medium (keratinocyte growth medium, KGM) was increased from 0.05 mm (KGM-L) to 1.2 mm (KGM-H). Here we examined the mechanism underlying this Ca2+-dependent generation of the acinar cell phenotype. Compared with cells in KGM-L, those in KGM-H display enhancement of Orai1, STIM1, STIM2, and nuclear factor of activated T cells 1 (NFAT1) expression together with an increase in store-operated Ca2+ entry (SOCE), SOCE-dependent nuclear translocation of pGFP-NFAT1, and NFAT-dependent but not NFκB-dependent gene expression. Importantly, AQP5, an acinar-specific protein critical for function, is up-regulated in KGM-H via SOCE/NFAT-dependent gene expression. We identified critical NFAT binding motifs in the AQP5 promoter that are involved in Ca2+-dependent up-regulation of AQP5. These important findings reveal that the Ca2+-induced switch of salivary epithelial cells to an acinar-like phenotype involves remodeling of SOCE and NFAT signaling, which together control the expression of proteins critically relevant for acinar cell function. Our data provide a novel strategy for generating and maintaining acinar cells in culture. PMID:26903518

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

NASA Astrophysics Data System (ADS)

Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

2015-02-01

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing β-Cell Apoptosis in Aging.

PubMed

Xiong, Yuyan; Yepuri, Gautham; Necetin, Sevil; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-06-01

Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L -arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic β-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II -/- ) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and β-cell mass, and more proliferative and less apoptotic β-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II -/- ) mice contains higher TNF-α levels than the young mice and stimulates β-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to β-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice. © 2017 by the American Diabetes Association.

The vacuole within

PubMed Central

Ellis, Kathryn; Hoffman, Brenton D.; Bagnat, Michel

2013-01-01

The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuoles results in a shortened embryo along the anterior-posterior axis. Interestingly, we discovered that notochord vacuoles are also essential for proper spine morphogenesis and that vacuole defects lead to scoliosis of the spine. Here we discuss the cellular organization of the notochord and how key features of its architecture allow the notochord to function in embryonic axis elongation and spine formation. PMID:23887209

Functional somatostatin receptors on a rat pancreatic acinar cell line

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

1988-07-01

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

Pancreatic acinar cell carcinoma extending into the common bile and main pancreatic ducts.

PubMed

Yamaguchi, Rin; Okabe, Yoshinobu; Jimi, Atsuo; Shiota, Koji; Kodama, Takahito; Naito, Yoshiki; Yasunaga, Masafumi; Kinoshita, Hisafumi; Kojiro, Masamichi

2006-10-01

Acinar cell carcinoma (ACC) of the pancreas is relatively rare, accounting for only approximately 1% of all exocrine pancreatic tumors. A 69-year-old man was found to have a mass lesion measuring approximately 4 cm in diameter in the pancreatic head on ultrasound, abdominal dynamic CT, and percutaneous transhepatic cholangiography. Magnetic resonance cholangiopancreatography showed defect of the lower common bile duct (CBD) due to obstruction by the tumor cast. Histopathologically, the pancreatic head tumor invaded the main pancreatic duct (MPD) and CBD with extension into the CBD in a form of tumor cast. The tumor cells consisted of a solid proliferation with abundant eosinophilic cytoplasm and round nuclei in an acinar and trabecular fashion. A 55-year-old man with upper abdominal pain and nausea, had a cystic lesion approximately 3 cm in size in the pancreatic tail on CT. Histopathologically, the tumor was encapsulated by fibrous capsule and had extensive central necrosis with solid areas in the tumor periphery, and invaded with extension into the MPD in a form of tumor cast. The tumor cells resembled acinar cells in solid growths. Two resected cases of ACC with unusual tumor extension into the CBD and the MPD, respectively, are reported.

Calcium signalling in the acinar environment of the exocrine pancreas: physiology and pathophysiology.

PubMed

Gryshchenko, Oleksiy; Gerasimenko, Julia V; Peng, Shuang; Gerasimenko, Oleg V; Petersen, Ole H

2018-02-09

Ca 2+ signalling in different cell types in exocrine pancreatic lobules was monitored simultaneously and signalling responses to various stimuli were directly compared. Ca 2+ signals evoked by K + -induced depolarization were recorded from pancreatic nerve cells. Nerve cell stimulation evoked Ca 2+ signals in acinar but not in stellate cells. Stellate cells are not electrically excitable as they, like acinar cells, did not generate Ca 2+ signals in response to membrane depolarization. The responsiveness of the stellate cells to bradykinin was markedly reduced in experimental alcohol-related acute pancreatitis, but they became sensitive to stimulation with trypsin. Our results provide fresh evidence for an important role of stellate cells in acute pancreatitis. They seem to be a critical element in a vicious circle promoting necrotic acinar cell death. Initial trypsin release from a few dying acinar cells generates Ca 2+ signals in the stellate cells, which then in turn damage more acinar cells causing further trypsin liberation. Physiological Ca 2+ signals in pancreatic acinar cells control fluid and enzyme secretion, whereas excessive Ca 2+ signals induced by pathological agents induce destructive processes leading to acute pancreatitis. Ca 2+ signals in the peri-acinar stellate cells may also play a role in the development of acute pancreatitis. In this study, we explored Ca 2+ signalling in the different cell types in the acinar environment of the pancreatic tissue. We have, for the first time, recorded depolarization-evoked Ca 2+ signals in pancreatic nerves and shown that whereas acinar cells receive a functional cholinergic innervation, there is no evidence for functional innervation of the stellate cells. The stellate, like the acinar, cells are not electrically excitable as they do not generate Ca 2+ signals in response to membrane depolarization. The principal agent evoking Ca 2+ signals in the stellate cells is bradykinin, but in experimental alcohol

Activating transcription factor 3 promotes loss of the acinar cell phenotype in response to cerulein-induced pancreatitis in mice.

PubMed

Fazio, Elena N; Young, Claire C; Toma, Jelena; Levy, Michael; Berger, Kurt R; Johnson, Charis L; Mehmood, Rashid; Swan, Patrick; Chu, Alphonse; Cregan, Sean P; Dilworth, F Jeffrey; Howlett, Christopher J; Pin, Christopher L

2017-09-01

Pancreatitis is a debilitating disease of the exocrine pancreas that, under chronic conditions, is a major susceptibility factor for pancreatic ductal adenocarcinoma (PDAC). Although down-regulation of genes that promote the mature acinar cell fate is required to reduce injury associated with pancreatitis, the factors that promote this repression are unknown. Activating transcription factor 3 (ATF3) is a key mediator of the unfolded protein response, a pathway rapidly activated during pancreatic insult. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that ATF3 is bound to the transcriptional regulatory regions of >30% of differentially expressed genes during the initiation of pancreatitis. Of importance, ATF3-dependent regulation of these genes was observed only upon induction of pancreatitis, with pathways involved in inflammation, acinar cell differentiation, and cell junctions being specifically targeted. Characterizing expression of transcription factors that affect acinar cell differentiation suggested that acinar cells lacking ATF3 maintain a mature cell phenotype during pancreatitis, a finding supported by maintenance of junctional proteins and polarity markers. As a result, Atf3 -/- pancreatic tissue displayed increased tissue damage and inflammatory cell infiltration at early time points during injury but, at later time points, showed reduced acinar-to-duct cell metaplasia. Thus our results reveal a critical role for ATF3 as a key regulator of the acinar cell transcriptional response during injury and may provide a link between chronic pancreatitis and PDAC. © 2017 Fazio et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Ultrastructural and biochemical studies of the effect of polychlorinated biphenyl on mouse parotid gland cells.

PubMed

Kawasaki, G; Mataki, S; Mizuno, A

1995-01-01

These effects of polychlorinated biphenyl (PCB) were examined by light and electron microscopy and biochemical analysis of lysosomal enzyme activities. Several experimental protocols with dosage schedules of either 0.2, 2.0, or 20 mg/kg of PCB were used. Typical histological changes were observed in mice given 2 mg/kg of PCB in a single injection. There were no remarkable changes until 4 days after PCB administration; marked cytoplasmic vacuolation was observed in parotid acinar cells at 7 days. The activities of lysosomal enzymes increased after the PCB injection and their maximum values appeared consistently at 4 days after the treatment; the increases were threefold for acid phosphatase, twofold for beta-glucuronidase, threefold for cathepsin D, fivefold for cathepsin H and twofold for cathepsin L. As vacuolation was preceded by a large increase in lysosomal enzyme activities and the vacuoles co-localized with lysosomes, it is suggested that an increase in these activities induced by PCB may be closely related to the development of vacuolation in the parotid acinar cells as a subacute effect of PCB.

The nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis.

PubMed

Hale, Michael A; Swift, Galvin H; Hoang, Chinh Q; Deering, Tye G; Masui, Toshi; Lee, Youn-Kyoung; Xue, Jumin; MacDonald, Raymond J

2014-08-01

The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype. © 2014. Published by The Company of Biologists Ltd.

Characterization of synthesis and storage of TGF-alpha in rat parotid acinar and intercalated duct cells.

PubMed

Login, G R; Yang, J; Bryan, K P; Digenis, E C; McBride, J; Elovic, A; Quissell, D O; Dvorak, A M; Wong, D T

1997-03-01

Although the expression and biological role of transforming growth factor-alpha (TGF-alpha) have been explored in a variety of normal cells in mammalian species, little is known about the storage of TGF-alpha in secretory cells of exocrine organs. Parotid glands from four rats were homogenized for RNA isolation followed by reverse transcription-polymerase chain reaction to determine the presence of TGF-alpha message. In situ hybridization using a hamster-specific TGF-alpha riboprobe was done on paraffin sections. Parotid gland and isolated acinar cells were processed for transmission electron microscopy (TEM) and postembedding immunogold labeled for TGF-alpha. Gold particles were counted on approximately 200 granules in 10 acinar cells and in 10 intercalated duct cells. Labeling density was calculated as the number of gold particles per square micrometer +/- SD. Statistical significance was calculated using one-way analysis of variance. Using multiple technologies, we have established that rat parotid acinar and intercalated duct cells synthesize TGF-alpha and store the precursor form of this cytokine in their secretory granules.

ptf1a+ , ela3l- cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae.

PubMed

Schmitner, Nicole; Kohno, Kenji; Meyer, Dirk

2017-03-01

The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l- negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l -positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b In conclusion, we

ptf1a+, ela3l− cells are developmentally maintained progenitors for exocrine regeneration following extreme loss of acinar cells in zebrafish larvae

PubMed Central

Schmitner, Nicole; Kohno, Kenji

2017-01-01

ABSTRACT The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l-negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l-positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b. In

Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

NASA Astrophysics Data System (ADS)

Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

2005-04-01

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

Functional differences in the acinar cells of the murine major salivary glands.

PubMed

Kondo, Y; Nakamoto, T; Jaramillo, Y; Choi, S; Catalan, M A; Melvin, J E

2015-05-01

In humans, approximately 90% of saliva is secreted by the 3 major salivary glands: the parotid (PG), the submandibular (SMG), and the sublingual glands (SLG). Even though it is known that all 3 major salivary glands secrete saliva by a Cl(-)-dependent mechanism, salivary secretion rates differ greatly among these glands. The goal of this study was to gain insight into the properties of the ion-transporting pathways in acinar cells that might account for the differences among the major salivary glands. Pilocarpine-induced saliva was simultaneously collected in vivo from the 3 major salivary glands of mice. When normalized by gland weight, the amount of saliva secreted by the PG was more than 2-fold larger than that obtained from the SMG and SLG. At the cellular level, carbachol induced an increase in the intracellular [Ca(2+)] that was more than 2-fold larger in PG and SMG than in SLG acinar cells. Carbachol-stimulated Cl(-) efflux and the protein levels of the Ca(2+)-activated Cl(-) channel TMEM16A, the major apical Cl(-) efflux pathway in salivary acinar cells, were significantly greater in PG compared with SMG and SLG. In addition, we evaluated the transporter activity of the Na(+)-K(+)-2Cl(-) cotransporters (NKCC1) and anion exchangers (AE), the 2 primary basolateral Cl(-) uptake mechanisms in acinar cells. The SMG NKCC1 activity was about twice that of the PG and more than 12-fold greater than that of the SLG. AE activity was similar in PG and SLG, and both PG and SLG AE activity was about 2-fold larger than that of SMG. In summary, the salivation kinetics of the 3 major glands are distinct, and these differences can be explained by the unique functional properties of each gland related to Cl(-) movement, including the transporter activities of the Cl(-) uptake and efflux pathways, and intracellular Ca(2+) mobilization. © International & American Associations for Dental Research 2015.

Establishment of Functional Acinar-like Cultures from Human Salivary Glands

PubMed Central

Jang, S.I.; Ong, H.L.; Gallo, A.; Liu, X.; Illei, G.

2015-01-01

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers—namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients. PMID:25416669

Establishment of functional acinar-like cultures from human salivary glands.

PubMed

Jang, S I; Ong, H L; Gallo, A; Liu, X; Illei, G; Alevizos, I

2015-02-01

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers-namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients. © International & American Associations for Dental Research 2014.

Age-related alterations in IL-1beta, TNF-alpha, and IL-6 concentrations in parotid acinar cells from BALB/c and non-obese diabetic mice.

PubMed

Yamakawa, M; Weinstein, R; Tsuji, T; McBride, J; Wong, D T; Login, G R

2000-08-01

IL-1beta, TNF-alpha, and IL-6 have been implicated in the destruction of parotid gland acinar cells (but not duct cells) in autoimmune sialoadenitis. Here we report the temporal alterations of these cytokines in parotid acinar cells that may lead to this specificity in cell death in the non-obese diabetic (NOD) mouse model for Sjögren's syndrome. Immunohistochemistry on paraffin sections of parotid gland from 5- and 10-week-old BALB/c and NOD mice confirmed the presence of many peri-acinar lymphoid nodules but few T-cells and macrophages between acinar cells. RT-PCR on enzymatically dispersed mouse parotid acinar cells (MPACs) showed no bands for CD3varepsilon, CD20, or F4/80 regardless of mouse strain or age. By ELISA, MPACs from 10-week-old NODs showed a small but highly significant (p<0.003) increase in IL-1beta and a large significant decrease (p<0.008) in IL-6 compared to 5-week-old NODs. Norepinephrine-stimulated amylase release from MPACs was not different regardless of mouse strain or age. These data show that alterations in acinar cell production of IL-1beta and IL-6 in aging NODs precede periductal lymphoid aggregates and acinar cell secretory dysfunction. (J Histochem Cytochem 48:1033-1041,2000)

Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

PubMed

van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

2015-04-01

Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.

PubMed

Dorrell, Craig; Grompe, Maria T; Pan, Fong Cheng; Zhong, Yongping; Canaday, Pamela S; Shultz, Leonard D; Greiner, Dale L; Wright, Chris V; Streeter, Philip R; Grompe, Markus

2011-06-06

Tools permitting the isolation of live pancreatic cell subsets for culture and/or molecular analysis are limited. To address this, we developed a collection of monoclonal antibodies with selective surface labeling of endocrine and exocrine pancreatic cell types. Cell type labeling specificity and cell surface reactivity were validated on mouse pancreatic sections and by gene expression analysis of cells isolated using FACS. Five antibodies which marked populations of particular interest were used to isolate and study viable populations of purified pancreatic ducts, acinar cells, and subsets of acinar cells from whole pancreatic tissue or of alpha or beta cells from isolated mouse islets. Gene expression analysis showed the presence of known endocrine markers in alpha and beta cell populations and revealed that TTR and DPPIV are primarily expressed in alpha cells whereas DGKB and GPM6A have a beta cell specific expression profile. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

PubMed Central

Huang, Ze-bing; Wang, Hai-yan; Sun, Na-na; Wang, Jing-ke; Zhao, Meng-qin; Shen, Jian-xin; Gao, Ming; Hammer, Ronald P; Fan, Xue-gong; Wu, Jie

2014-01-01

Aim: Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca2+ oscillations in mouse pancreatic acinar cells in vitro. Methods: Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca2+ oscillations were monitored by whole-cell recording of Ca2+-activated Cl− currents and by using confocal Ca2+ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established. Results: Bath application of ACh (10 nmol/L) induced typical Ca2+ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca2+ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca2+ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca2+ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca2+ oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca2+ oscillations. Conclusion: Congo red significantly modulates intracellular Ca2+ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug. PMID:25345744

Pancreatic panniculitis as a presentation symptom of acinar cell carcinoma.

PubMed

de Frutos Rosa, Diego; Espinosa Taranilla, Laura; González de Canales de Simón, Pilar; Vélez Velázquez, María Dolores; Guirado Koch, Cristina

2018-05-01

Pancreatic panniculitis is a rare skin manifestation associated with pancreatic conditions. This condition has similar characteristics to those of other panniculitis types and its course parallels the triggering condition and may occasionally precede it. We report the case of a female patient with asymptomatic pancreatic panniculitis; the etiologic study identified a pancreatic acinar cell carcinoma with liver metastases.

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

PubMed Central

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-01-01

Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

PubMed Central

Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

2015-01-01

Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

PubMed Central

Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

Adenovirus-mediated hAQP1 expression in irradiated mouse salivary glands causes recovery of saliva secretion by enhancing acinar cell volume decrease

PubMed Central

Teos, LY; Zheng, C-Y; Liu, X; Swaim, WD; Goldsmith, CM; Cotrim, AP; Baum, BJ; Ambudkar, IS

2017-01-01

Head and neck irradiation (IR) during cancer treatment causes by-stander effects on the salivary glands leading to irreversible loss of saliva secretion. The mechanism underlying loss of fluid secretion is not understood and no adequate therapy is currently available. Delivery of an adenoviral vector encoding human aquaporin-1 (hAQP1) into the salivary glands of human subjects and animal models with radiation-induced salivary hypofunction leads to significant recovery of saliva secretion and symptomatic relief in subjects. To elucidate the mechanism underlying loss of salivary secretion and the basis for AdhAQP1-dependent recovery of salivary gland function we assessed submandibular gland function in control mice and mice 2 and 8 months after treatment with a single 15-Gy dose of IR (delivered to the salivary gland region). Salivary secretion and neurotransmitter-stimulated changes in acinar cell volume, an in vitro read-out for fluid secretion, were monitored. Consistent with the sustained 60% loss of fluid secretion following IR, a carbachol (CCh)-induced decrease in acinar cell volume from the glands of mice post IR was transient and attenuated as compared with that in cells from non-IR age-matched mice. The hAQP1 expression in non-IR mice induced no significant effect on salivary fluid secretion or CCh-stimulated cell volume changes, except in acinar cells from 8-month group where the initial rate of cell shrinkage was increased. Importantly, the expression of hAQP1 in the glands of mice post IR induced recovery of salivary fluid secretion and a volume decrease in acinar cells to levels similar to those in cells from non-IR mice. The initial rates of CCh-stimulated cell volume reduction in acinar cells from hAQP1-expressing glands post IR were similar to those from control cells. Altogether, the data suggest that expression of hAQP1 increases the water permeability of acinar cells, which underlies the recovery of fluid secretion in the salivary glands

Docosahexaenoic acid inhibits IL-6 expression via PPARγ-mediated expression of catalase in cerulein-stimulated pancreatic acinar cells.

PubMed

Song, Eun Ah; Lim, Joo Weon; Kim, Hyeyoung

2017-07-01

Cerulein pancreatitis mirrors human acute pancreatitis. In pancreatic acinar cells exposed to cerulein, reactive oxygen species (ROS) mediate inflammatory signaling by Janus kinase (JAK) 2/signal transducer and activator of transcription (STAT) 3, and cytokine induction. Docosahexaenoic acid (DHA) acts as an agonist of peroxisome proliferator activated receptor γ (PPARγ), which mediates the expression of some antioxidant enzymes. We hypothesized that DHA may induce PPARγ-target catalase expression and reduce ROS levels, leading to the inhibition of JAK2/STAT3 activation and IL-6 expression in cerulein-stimulated acinar cells. Pancreatic acinar AR42J cells were treated with DHA in the presence or absence of the PPARγ antagonist GW9662, or treated with the PPARγ agonist troglitazone, and then stimulated with cerulein. Expression of IL-6 and catalase, ROS levels, JAK2/STAT3 activation, and nuclear translocation of PPARγ were assessed. DHA suppressed the increase in ROS, JAK2/STAT3 activation, and IL-6 expression induced nuclear translocation of PPARγ and catalase expression in cerulein-stimulated AR42J cells. Troglitazone inhibited the cerulein-induced increase in ROS and IL-6 expression, but induced catalase expression similar to DHA in AR42J cells. GW9662 abolished the inhibitory effect of DHA on cerulein-induced increase in ROS and IL-6 expression in AR42J cells. DHA-induced expression of catalase was suppressed by GW9662 in cerulein-stimulated AR42J cells. Thus, DHA induces PPARγ activation and catalase expression, which inhibits ROS-mediated activation of JAK2/STAT3 and IL-6 expression in cerulein-stimulated pancreatic acinar cells. Copyright © 2017. Published by Elsevier Ltd.

Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells.

PubMed

Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

2012-07-01

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25-30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10(-6) M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells

PubMed Central

Stępiński, D.

2012-01-01

In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system. PMID:22688294

Biochemical analysis of secretory proteins synthesized by normal rat pancreas and by pancreatic acinar tumor cells

PubMed Central

1982-01-01

We have examined the secretogogue responsiveness and the pattern of secretory proteins produced by a transplantable rat pancreatic acinar cell tumor. Dispersed tumor cells were found to discharge secretory proteins in vitro when incubated with hormones that act on four different classes of receptors: carbamylcholine, caerulein, secretin- vasoactive intestinal peptide, and bombesin. With all hormones tested, maximal discharge from tumor cells was only about one-half that of control pancreatic lobules, but occurred at the same dose optima except for secretin, whose dose optimum was 10-fold higher. Biochemical analysis of secretory proteins discharged by the tumor cells was carried out by crossed immunoelectrophoresis and by two-dimensional isoelectric focusing-SDS polyacrylamide gel electrophoresis. To establish a baseline for comparison, secretory proteins from normal rat pancreas were identified according to enzymatic activity and correlated with migration position on two-dimensional gels. Our results indicate that a group of basic polypeptides including proelastase, basic trypsinogen, basic chymotrypsinogen, and ribonuclease, two out of three forms of procarboxypeptidase B, and the major lipase species were greatly reduced or absent in tumor cell secretion. In contrast, the amount of acidic chymotrypsinogen was notably increased compared with normal acinar cells. Although the acinar tumor cells are highly differentiated cytologically and express functional receptors for several classes of pancreatic secretagogues, they show quantitative and qualitative differences when compared with normal pancreas with regard to their production of secretory proteins. PMID:6185502

Establishment and characterization of a new human acinar cell carcinoma cell line, Faraz-ICR, from pancreas.

PubMed

Rezaei, Marzieh; Hosseini, Ahmad; Nikeghbalian, Saman; Ghaderi, Abbas

Basic research in the field of acinar cell carcinoma (ACC) as a rare neoplasm of the pancreas is dependent on the availability of pragmatic model such as new pancreatic cancer cell lines. Thus, establishment and characterization of new pancreatic cancer cell lines from ACC origin are deemed important. Faraz-ICR cell line was derived from a 58-years old woman with pancreatic acinar cell carcinoma by the collagenase digestion protocol. We characterized the cell line by examining its morphology and cytostructural and functional profile. Faraz-ICR has a doubling time of 35 hours and grows in soft agar with a colony-forming efficiency of 25%. The cell had nearly normal pattern of chromosomes in karyotype analysis and Comparative Genomic Hybridization (CGH) array analysis. Evaluation of cells by flowcytometry showed that Faraz-ICR is negative for EpCAM and mesenchymal markers in different passages, and has epithelial nature. Immunofluorescence staining revealed that cells were strongly positive for vimentin, desmin, ezrin, S100, nestin and they were negative for pan-cytokeratins, chromogranin and alpha smooth muscle actin. We were able to establish a new pancreatic carcinoma cell line with partial aspects of Epithelial-mesenchymal transition and aggressiveness. This cell line might be suitable for studying various anticancer drugs and protein profile aiming to see any possible tumor associated marker for ACC. Copyright © 2017 IAP and EPC. Published by Elsevier B.V. All rights reserved.

RNA synthesis in the pancreatic acinar cells of aging mice as revealed by electron microscopic radioautography.

PubMed

Nagata, Tetsuji

2012-01-01

For the purpose of studying the aging changes of macromolecular synthesis in the pancreatic acinar cells of experimental animals, we studied 10 groups of aging mice during development and aging from fetal day 19 to postnatal month 24. They were injected with 3H-uridine, a precursor for RNA synthesis, sacrificed and the pancreatic tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating RNA synthesis in pancreatic acinar cells in respective aging groups were analyzed qualitatively. The number of mitochondria per cell, the number of labeled mitochondria with silver grains and the number of silver grains in each cell in respective aging groups were analyzed quantitatively in relation to the aging of animals. The results revealed that the RNA synthetic activity as expressed by the incorporations of RNA precursor, i.e., the number of silver grains in cell nuclei, cell organelles, changed due to the aging of animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each pancreatic acinar cell. It was demonstrated that the number of mitochondria, the number of labeled mitochondria and the labeling indices showing RNA synthesis at various ages increased from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile stage at postnatal year 1 to 2, indicating the aging changes. Based upon our findings, available literature on macromolecular synthesis in mitochondria of various cells are reviewed.

Assessing the secretory capacity of pancreatic acinar cells.

PubMed

Geron, Erez; Schejter, Eyal D; Shilo, Ben-Zion

2014-08-28

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells.

PubMed

Brown, David A; Melvin, James E; Yule, David I

2003-11-01

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

Sulforaphane Protects Pancreatic Acinar Cell Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3 Inflammatory Pathway

PubMed Central

Dong, Zhaojun; Shang, Haixiao; Chen, Yong Q.; Pan, Li-Long

2016-01-01

Acute pancreatitis (AP) is characterized by early activation of intra-acinar proteases followed by acinar cell death and inflammation. Cellular oxidative stress is a key mechanism underlying these pathological events. Sulforaphane (SFN) is a natural organosulfur antioxidant with undescribed effects on AP. Here we investigated modulatory effects of SFN on cellular oxidation and inflammation in AP. AP was induced by cerulean hyperstimulation in BALB/c mice. Treatment group received a single dose of 5 mg/kg SFN for 3 consecutive days before AP. We found that SFN administration attenuated pancreatic injury as evidenced by serum amylase, pancreatic edema, and myeloperoxidase, as well as by histological examination. SFN administration reverted AP-associated dysregulation of oxidative stress markers including pancreatic malondialdehyde and redox enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). In acinar cells, SFN treatment upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes including quinoneoxidoreductase-1, heme oxidase-1, SOD1, and GPx1. In addition, SFN selectively suppressed cerulein-induced activation of the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome, in parallel with reduced nuclear factor- (NF-) κB activation and modulated NF-κB-responsive cytokine expression. Together, our data suggested that SFN modulates Nrf2-mediated oxidative stress and NLRP3/NF-κB inflammatory pathways in acinar cells, thereby protecting against AP. PMID:27847555

Sulforaphane Protects Pancreatic Acinar Cell Injury by Modulating Nrf2-Mediated Oxidative Stress and NLRP3 Inflammatory Pathway.

PubMed

Dong, Zhaojun; Shang, Haixiao; Chen, Yong Q; Pan, Li-Long; Bhatia, Madhav; Sun, Jia

2016-01-01

Acute pancreatitis (AP) is characterized by early activation of intra-acinar proteases followed by acinar cell death and inflammation. Cellular oxidative stress is a key mechanism underlying these pathological events. Sulforaphane (SFN) is a natural organosulfur antioxidant with undescribed effects on AP. Here we investigated modulatory effects of SFN on cellular oxidation and inflammation in AP. AP was induced by cerulean hyperstimulation in BALB/c mice. Treatment group received a single dose of 5 mg/kg SFN for 3 consecutive days before AP. We found that SFN administration attenuated pancreatic injury as evidenced by serum amylase, pancreatic edema, and myeloperoxidase, as well as by histological examination. SFN administration reverted AP-associated dysregulation of oxidative stress markers including pancreatic malondialdehyde and redox enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx). In acinar cells, SFN treatment upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes including quinoneoxidoreductase-1, heme oxidase-1, SOD1, and GPx1. In addition, SFN selectively suppressed cerulein-induced activation of the nucleotide-binding domain leucine-rich repeat containing family, pyrin domain-containing 3 (NLRP3) inflammasome, in parallel with reduced nuclear factor- (NF-) κ B activation and modulated NF- κ B-responsive cytokine expression. Together, our data suggested that SFN modulates Nrf2-mediated oxidative stress and NLRP3/NF- κ B inflammatory pathways in acinar cells, thereby protecting against AP.

The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma

PubMed Central

Krah, Nathan M; De La O, Jean-Paul; Swift, Galvin H; Hoang, Chinh Q; Willet, Spencer G; Chen Pan, Fong; Cash, Gabriela M; Bronner, Mary P; Wright, Christopher VE; MacDonald, Raymond J; Murtaugh, L Charles

2015-01-01

Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas. DOI: http://dx.doi.org/10.7554/eLife.07125.001 PMID:26151762

A Single Injection of Interleukin-1 Induces Reversible Aqueous-tear Deficiency, Lacrimal Gland Inflammation, and Acinar and Ductal Cell Proliferation

PubMed Central

Zoukhri, Driss; Macari, Elizabeth; Kublin, Claire L.

2011-01-01

Emerging studies from our laboratory demonstrate that interleukin-1 (IL-1) family members play a major role in impairing lacrimal gland functions. Here we have extended our investigations to observe the effects of IL-1 on aqueous tear production, lacrimal gland secretion, lacrimal gland histology, and acinar and ductal cell proliferation. We demonstrate that a single injection of IL-1 into the lacrimal glands inhibited neurally- as well as agonist-induced protein secretion resulting in decreased tear output. Meanwhile, IL-1 injection induced a severe, but reversible (7–13 days), inflammatory response that led to destruction of lacrimal gland acinar epithelial cells. Finally, we demonstrate that as the inflammatory response subsided and lacrimal gland secretion and tear production returned to normal levels, there was increased proliferation of acinar and ductal epithelial cells. Our work uncovers novel effects of IL-1 on lacrimal gland functions and the potential regenerative capacity of the mouse lacrimal gland. PMID:17362931

Nicotine promotes initiation and progression of KRAS-induced pancreatic cancer via Gata6-dependent dedifferentiation of acinar cells in mice.

PubMed

Hermann, Patrick C; Sancho, Patricia; Cañamero, Marta; Martinelli, Paola; Madriles, Francesc; Michl, Patrick; Gress, Thomas; de Pascual, Ricardo; Gandia, Luis; Guerra, Carmen; Barbacid, Mariano; Wagner, Martin; Vieira, Catarina R; Aicher, Alexandra; Real, Francisco X; Sainz, Bruno; Heeschen, Christopher

2014-11-01

Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting

New saliva secretion model based on the expression of Na+-K+ pump and K+ channels in the apical membrane of parotid acinar cells.

PubMed

Almássy, János; Siguenza, Elias; Skaliczki, Marianna; Matesz, Klara; Sneyd, James; Yule, David I; Nánási, Péter P

2018-04-01

The plasma membrane of parotid acinar cells is functionally divided into apical and basolateral regions. According to the current model, fluid secretion is driven by transepithelial ion gradient, which facilitates water movement by osmosis into the acinar lumen from the interstitium. The osmotic gradient is created by the apical Cl - efflux and the subsequent paracellular Na + transport. In this model, the Na + -K + pump is located exclusively in the basolateral membrane and has essential role in salivary secretion, since the driving force for Cl - transport via basolateral Na + -K + -2Cl - cotransport is generated by the Na + -K + pump. In addition, the continuous electrochemical gradient for Cl - flow during acinar cell stimulation is maintained by the basolateral K + efflux. However, using a combination of single-cell electrophysiology and Ca 2+ -imaging, we demonstrate that photolysis of Ca 2+ close to the apical membrane of parotid acinar cells triggered significant K + current, indicating that a substantial amount of K + is secreted into the lumen during stimulation. Nevertheless, the K + content of the primary saliva is relatively low, suggesting that K + might be reabsorbed through the apical membrane. Therefore, we investigated the localization of Na + -K + pumps in acinar cells. We show that the pumps appear evenly distributed throughout the whole plasma membrane, including the apical pole of the cell. Based on these results, a new mathematical model of salivary fluid secretion is presented, where the pump reabsorbs K + from and secretes Na + to the lumen, which can partially supplement the paracellular Na + pathway.

Snail1 is required for the maintenance of the pancreatic acinar phenotype

PubMed Central

Loubat-Casanovas, Jordina; Peña, Raúl; Gonzàlez, Núria; Alba-Castellón, Lorena; Rosell, Santi; Francí, Clara; Navarro, Pilar; de Herreros, Antonio García

2016-01-01

The Snail1 transcriptional factor is required for correct embryonic development, yet its expression in adult animals is very limited and its functional roles are not evident. We have now conditionally inactivated Snail1 in adult mice and analyzed the phenotype of these animals. Snail1 ablation rapidly altered pancreas structure: one month after Snail1 depletion, acinar cells were markedly depleted, and pancreas accumulated adipose tissue. Snail1 expression was not detected in the epithelium but was in pancreatic mesenchymal cells (PMCs). Snail1 ablation in cultured PMCs downregulated the expression of several β-catenin/Tcf-4 target genes, modified the secretome of these cells and decreased their ability to maintain acinar markers in cultured pancreas cells. Finally, Snail1 deficiency modified the phenotype of pancreatic tumors generated in transgenic mice expressing c-myc under the control of the elastase promoter. Specifically, Snail1 depletion did not significantly alter the size of the tumors but accelerated acinar-ductal metaplasia. These results demonstrate that Snail1 is expressed in PMCs and plays a pivotal role in maintaining acinar cells within the pancreas in normal and pathological conditions. PMID:26735179

Cellular vacuolation induced by Clostridium perfringens epsilon-toxin.

PubMed

Nagahama, Masahiro; Itohayashi, Yukari; Hara, Hideki; Higashihara, Masahiro; Fukatani, Yusuke; Takagishi, Teruhisa; Oda, Masataka; Kobayashi, Keiko; Nakagawa, Ichiro; Sakurai, Jun

2011-09-01

The epsilon-toxin of Clostridium perfringens forms a heptamer in the membranes of Madin-Darby canine kidney cells, leading to cell death. Here, we report that it caused the vacuolation of Madin-Darby canine kidney cells. The toxin induced vacuolation in a dose-dependent and time-dependent manner. The monomer of the toxin formed oligomers on lipid rafts in membranes of the cells. Methyl-β-cyclodextrin and poly(ethylene glycol) 4000 inhibited the vacuolation. Epsilon-toxin was internalized into the cells. Confocal microscopy revealed that the internalized toxin was transported from early endosomes (early endosome antigen 1 staining) to late endosomes and lysosomes (lysosomal-associated membrane protein 2 staining) and then distributed to the membranes of vacuoles. Furthermore, the vacuolation was inhibited by bafilomycin A1, a V-type ATPase inhibitor, and colchicine and nocodazole, microtubule-depolymerizing agents. The early endosomal marker green fluorescent protein-Rab5 and early endosome antigen 1 did not localize to vacuolar membranes. In contrast, the vacuolar membranes were specifically stained by the late endosomal and lysosomal marker green fluorescent protein-Rab7 and lysosomal-associated membrane protein 2. The vacuoles in the toxin-treated cells were stained with LysoTracker Red DND-99, a marker for late endosomes and lysosomes. A dominant negative mutant of Rab7 prevented the vacuolization, whereas a mutant form of Rab5 was less effective. These results demonstrate, for the first time, that: (a) oligomers of epsilon-toxin formed in lipid rafts are endocytosed; and (b) the vacuoles originating from late endosomes and lysosomes are formed by an oligomer of epsilon-toxin. © 2011 The Authors Journal compilation © 2011 FEBS.

Sirtuin-1 regulates acinar-to-ductal metaplasia and supports cancer cell viability in pancreatic cancer.

PubMed

Wauters, Elke; Sanchez-Arévalo Lobo, Victor J; Pinho, Andreia V; Mawson, Amanda; Herranz, Daniel; Wu, Jianmin; Cowley, Mark J; Colvin, Emily K; Njicop, Erna Ngwayi; Sutherland, Rob L; Liu, Tao; Serrano, Manuel; Bouwens, Luc; Real, Francisco X; Biankin, Andrew V; Rooman, Ilse

2013-04-01

The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD(+)-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. ©2012 AACR.

Gata6 is required for complete acinar differentiation and maintenance of the exocrine pancreas in adult mice.

PubMed

Martinelli, Paola; Cañamero, Marta; del Pozo, Natalia; Madriles, Francesc; Zapata, Agustín; Real, Francisco X

2013-10-01

Previous studies have suggested an important role of the transcription factor Gata6 in endocrine pancreas, while GATA6 haploinsufficient inactivating mutations cause pancreatic agenesis in humans. We aimed to analyse the effects of Gata6 inactivation on pancreas development and function. We deleted Gata6 in all epithelial cells in the murine pancreas at the onset of its development. Acinar proliferation, apoptosis, differentiation and exocrine functions were assessed using reverse transcriptase quantitative PCR (RT-qPCR), chromatin immunoprecipitation, immunohistochemistry and enzyme assays. Adipocyte transdifferentiation was assessed using electron microscopy and genetic lineage tracing. Gata6 is expressed in all epithelial cells in the adult mouse pancreas but it is only essential for exocrine pancreas homeostasis: while dispensable for pancreatic development after e10.5, it is required for complete acinar differentiation, for establishment of polarity and for the maintenance of acinar cells in the adult. Gata6 regulates directly the promoter of genes coding for digestive enzymes and the transcription factors Rbpjl and Mist1. Upon pancreas-selective Gata6 inactivation, massive loss of acinar cells and fat replacement take place. This is accompanied by increased acinar apoptosis and proliferation, acinar-to-ductal metaplasia and adipocyte transdifferentiation. By contrast, the endocrine pancreas is spared. Our data show that Gata6 is required for the complete differentiation of acinar cells through multiple transcriptional regulatory mechanisms. In addition, it is required for the maintenance of the adult acinar cell compartment. Our studies suggest that GATA6 alterations may contribute to diseases of the human adult exocrine pancreas.

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

PubMed

Freedman, S D; Scheele, G A

1994-03-23

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell

PubMed Central

1995-01-01

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell

Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

NASA Astrophysics Data System (ADS)

Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

2008-02-01

Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

Effects of Ghrelin miRNA on Inflammation and Calcium Pathway in Pancreatic Acinar Cells of Acute Pancreatitis.

PubMed

Tang, Xiping; Tang, Guodu; Liang, Zhihai; Qin, Mengbin; Fang, Chunyun; Zhang, Luyi

The study investigated the effects of endogenous targeted inhibition of ghrelin gene on inflammation and calcium pathway in an in vitro pancreatic acinar cell model of acute pancreatitis. Lentiviral expression vector against ghrelin gene was constructed and transfected into AR42J cells. The mRNA and protein expression of each gene were detected by reverse transcription polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. The concentration of intracellular calcium ([Ca]i) was determined by calcium fluorescence mark probe combined with laser scanning confocal microscopy. Compared with the control group, cerulein could upregulate mRNA and protein expression of inflammatory factors, calcium pathway, ghrelin, and [Ca]i. mRNA and protein expression of inflammatory factors increased significantly in cells transfected with ghrelin miRNA compared with the other groups. Intracellular calcium and expression of some calcium pathway proteins decreased significantly in cells transfected with ghrelin miRNA compared with the other groups. Targeted inhibition of ghrelin gene in pancreatic acinar cells of acute pancreatitis can upregulate the expression of the intracellular inflammatory factors and alleviate the intracellular calcium overload.

Hydrogen sulfide: a novel gaseous signaling molecule and intracellular Ca2+ regulator in rat parotid acinar cells.

PubMed

Moustafa, Amira; Habara, Yoshiaki

2015-10-01

In addition to nitric oxide (NO), hydrogen sulfide (H2S) is recognized as a crucial gaseous messenger that exerts many biological actions in various tissues. An attempt was made to assess the roles and underlying mechanisms of both gases in isolated rat parotid acinar cells. Ductal cells and some acinar cells were found to express NO and H2S synthases. Cevimeline, a muscarinic receptor agonist upregulated endothelial NO synthase in parotid tissue. NO and H2S donors increased the intracellular Ca(2+) concentration ([Ca(2+)]i). This was not affected by inhibitors of phospholipase C and inositol 1,4,5-trisphosphate receptors, but was decreased by blockers of ryanodine receptors (RyRs), soluble guanylyl cyclase, and protein kinase G. The H2S donor evoked NO production, which was decreased by blockade of NO synthases or phosphoinositide 3-kinase or by hypotaurine, an H2S scavenger. The H2S donor-induced [Ca(2+)]i increase was diminished by a NO scavenger or the NO synthases blocker. These results suggest that NO and H2S play important roles in regulating [Ca(2+)]i via soluble guanylyl cyclase-cGMP-protein kinase G-RyRs, but not via inositol 1,4,5-trisphosphate receptors. The effect of H2S may be partially through NO produced via phosphoinositide 3-kinase-Akt-endothelial NO synthase. It was concluded that both gases regulate [Ca(2+)]i in a synergistic way, mainly via RyRs in rat parotid acinar cells. Copyright © 2015 the American Physiological Society.

Characteristics of weak base-induced vacuoles formed around individual acidic organelles.

PubMed

Hiruma, Hiromi; Kawakami, Tadashi

2011-01-01

We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.

Whole exome sequencing reveals recurrent mutations in BRCA2 and FAT genes in acinar cell carcinomas of the pancreas.

PubMed

Furukawa, Toru; Sakamoto, Hitomi; Takeuchi, Shoko; Ameri, Mitra; Kuboki, Yuko; Yamamoto, Toshiyuki; Hatori, Takashi; Yamamoto, Masakazu; Sugiyama, Masanori; Ohike, Nobuyuki; Yamaguchi, Hiroshi; Shimizu, Michio; Shibata, Noriyuki; Shimizu, Kyoko; Shiratori, Keiko

2015-03-06

Acinar cell carcinoma of the pancreas is a rare tumor with a poor prognosis. Compared to pancreatic ductal adenocarcinoma, its molecular features are poorly known. We studied a total of 11 acinar cell carcinomas, including 3 by exome and 4 by target sequencing. Exome sequencing revealed 65 nonsynonymous mutations and 22 indels with a mutation rate of 3.4 mutations/Mb per tumor, on average. By accounting for not only somatic but also germline mutations with loss of the wild-type allele, we identified recurrent mutations of BRCA2 and FAT genes. BRCA2 showed somatic or germline premature termination mutations, with loss of the wild-type allele in 3 of 7 tumors. FAT1, FAT3, and FAT4 showed somatic or germline missense mutations in 4 of 7 tumors. The germline FAT mutations were with loss of the wild-type allele. Loss of BRCA2 expression was observed in 5 of 11 tumors. One patient with a BRCA2-mutated tumor experienced complete remission of liver metastasis following cisplatinum chemotherapy. In conclusion, acinar cell carcinomas show a distinct mutation pattern and often harbor somatic or germline mutations of BRCA2 and FAT genes. This result may warrant assessment of BRCA2 abrogation in patients with the carcinoma to determine their sensitivity to chemotherapy.

Adjustment of Host Cells for Accommodation of Symbiotic Bacteria: Vacuole Defunctionalization, HOPS Suppression, and TIP1g Retargeting in Medicago[C][W][OPEN

PubMed Central

Gavrin, Aleksandr; Kaiser, Brent N.; Geiger, Dietmar; Tyerman, Stephen D.; Wen, Zhengyu; Bisseling, Ton; Fedorova, Elena E.

2014-01-01

In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria. PMID:25217511

Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development.

PubMed

Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

2012-01-15

During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3(+) progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3(CreERT/+)) and Neurog3-deficient (Neurog3(CreERT/CreERT)) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition, endocrine progenitor cells arise from bipotent precursors already committed to the duct/endocrine lineages and not from domain of cells having distinct potentialities. Copyright © 2011 Elsevier Inc. All rights reserved.

Competence of failed endocrine progenitors to give rise to acinar but not ductal cells is restricted to early pancreas development

PubMed Central

Beucher, Anthony; Martín, Mercè; Spenle, Caroline; Poulet, Martine; Collin, Caitlin; Gradwohl, Gérard

2011-01-01

SUMMARY During mouse pancreas development, the transient expression of Neurogenin3 (Neurog3) in uncommitted pancreas progenitors is required to determine endocrine destiny. However it has been reported that Neurog3-expressing cells can eventually adopt acinar or ductal fates and that Neurog3 levels were important to secure the islet destiny. It is not known whether the competence of Neurog3-induced cells to give rise to non-endocrine lineages is an intrinsic property of these progenitors or depends on pancreas developmental stage. Using temporal genetic labeling approaches we examined the dynamic of endocrine progenitor differentiation and explored the plasticity of Neurog3-induced cells throughout development. We found that Neurog3+ progenitors develop into hormone-expressing cells in a fast process taking less then 10h. Furthermore, fate-mapping studies in heterozygote (Neurog3CreERT/+) and Neurog3-deficient (Neurog3CreERT/CreERT) embryos revealed that Neurog3-induced cells have different potential over time. At the early bud stage, failed endocrine progenitors can adopt acinar or ductal fate, whereas later in the branching pancreas they do not contribute to the acinar lineage but Neurog3-deficient cells eventually differentiate into duct cells. Thus these results provide evidence that the plasticity of Neurog3-induced cells becomes restricted during development. Furthermore these data suggest that during the secondary transition endocrine progenitor cells arise from single bipotent progenitor already committed to the duct/endocrine lineages and not from domain of cells having both potentialities. PMID:22056785

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation.

PubMed

Wang, Feng; Gao, Zeng-Xin; Cai, Feng; Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

2017-08-22

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration.

A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture

PubMed Central

Jaquinod, Michel; Villiers, Florent; Kieffer-Jaquinod, Sylvie; Hugouvieux, Véronique; Bruley, Christophe; Garin, Jérôme; Bourguignon, Jacques

2007-01-01

To better understand the mechanisms governing cellular traffic, storage of various metabolites and their ultimate degradation, Arabidopsis thaliana vacuoles proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42 fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic study. Therefore, a proteomic approach was developed in order to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, (iii) a pre-fractionation of proteins by short migration on SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, 2/3 of which copurify with the membrane hydrophobic fraction and 1/3 with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were previously known to be associated with vacuolar

Changes in vacuolation in the root apex cells of soybean seedlings in microgravity

NASA Technical Reports Server (NTRS)

Klymchuk, D. O.; Kordyum, E. L.; Vorobyova, T. V.; Chapman, D. K.; Brown, C. S.

2003-01-01

Changes in the vacuolation in root apex cells of soybean (Glycine max L. [Merr.]) seedlings grown in microgravity were investigated. Spaceflight and ground control seedlings were grown in the absence or presence of KMnO4 (to remove ethylene) for 6 days. After landing, in order to study of cell ultrastructure and subcellular free calcium ion distribution, seedling root apices were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer and 2% (w/v) glutaraldehyde, 2.5% (w/v) formaldehyde, 2% (w/v) potassium antimonate K[Sb(OH)6] in 0.1 M K2HPO4 buffer with an osmolarity (calculated theoretically) of 0.45 and 1.26 osmol. The concentrations of ethylene in all spaceflight canisters were significantly higher than in the ground control canisters. Seedling growth was reduced in the spaceflight-exposed plants. Additionally, the spaceflight-exposed plants exhibited progressive vacuolation in the root apex cells, particularly in the columella cells, to a greater degree than the ground controls. Plasmolysis was observed in columella cells of spaceflight roots fixed in solutions with relatively high osmolarity (1.26 osmol). The appearance of plasmolysis permitted the evaluation of the water status of cells. The water potential of the spaceflight cells was higher than the surrounding fixative solution. A decrease in osmotic potential and/or an increase in turgor potential may have induced increases in cell water potential. However, the plasmolysed (i.e. non-turgid) cells implied that increases in water potential were accompanied with a decrease in osmotic potential. In such cells changes in vacuolation may have been involved to maintain turgor pressure or may have been a result of intensification of other vacuolar functions like digestion and storage. c2003 COSPAR. Published by Elsevier Ltd. All rights reserved.

Case report: primary acinar cell carcinoma of the liver treated with multimodality therapy

PubMed Central

Basturk, Olca; Shia, Jinru; Klimstra, David S.; Alago, William; D’Angelica, Michael I.; Abou-Alfa, Ghassan K.; O’Reilly, Eileen M.; Lowery, Maeve A.

2017-01-01

We describe a case of primary acinar cell carcinoma (ACC) originating in the liver in a 54-year-old female, diagnosed following persistent abnormal elevated liver function. Imaging revealed two masses, one dominant lesion in the right hepatic lobe and another in segment IVA. A right hepatectomy was performed to remove the larger lesion, while the mass in segment IVA was unresectable due to its proximity to the left hepatic vein. Immunohistochemical staining showed positivity for trypsin and chymotrypsin. Postoperatively the patient underwent hepatic arterial embolization of the other unresectable lesion followed by FOLFOX chemotherapy. At 20 months from diagnosis the patient is currently under observation with a decreasing necrotic mass and no other disease evident. Based on histology, immunohistochemistry and radiological findings a diagnosis of primary ACC of the liver was made. Genomic assessment of somatic mutations within the patient’s tumor was also performed through next generation sequencing and findings were consistent with an acinar malignancy. This case highlights a rare tumor subtype treated with a combination of therapeutic modalities through a multidisciplinary approach. PMID:29184698

Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

PubMed Central

Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

2017-01-01

Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

Neuronal Vacuolization in Feline Panleukopenia Virus Infection.

PubMed

Pfankuche, Vanessa M; Jo, Wendy K; van der Vries, Erhard; Jungwirth, Nicole; Lorenzen, Stephan; Osterhaus, Albert D M E; Baumgärtner, Wolfgang; Puff, Christina

2018-03-01

Feline panleukopenia virus (FPV) infections are typically associated with anorexia, vomiting, diarrhea, neutropenia, and lymphopenia. In cases of late prenatal or early neonatal infections, cerebellar hypoplasia is reported in kittens. In addition, single cases of encephalitis are described. FPV replication was recently identified in neurons, although it is mainly found in cells with high mitotic activity. A female cat, 2 months old, was submitted to necropsy after it died with neurologic deficits. Besides typical FPV intestinal tract changes, multifocal, randomly distributed intracytoplasmic vacuoles within neurons of the thoracic spinal cord were found histologically. Next-generation sequencing identified FPV-specific sequences within the central nervous system. FPV antigen was detected within central nervous system cells, including the vacuolated neurons, via immunohistochemistry. In situ hybridization confirmed the presence of FPV DNA within the vacuolated neurons. Thus, FPV should be considered a cause for neuronal vacuolization in cats presenting with ataxia.

Pancreatic Fat Accumulation, Fibrosis, and Acinar Cell Injury in the Zucker Diabetic Fatty Rat Fed a Chronic High-Fat Diet

PubMed Central

Matsuda, Akiko; Makino, Naohiko; Tozawa, Tomohiro; Shirahata, Nakao; Honda, Teiichiro; Ikeda, Yushi; Sato, Hideyuki; Ito, Miho; Kakizaki, Yasuharu; Akamatsu, Manabu; Ueno, Yoshiyuki; Kawata, Sumio

2014-01-01

Objective The histological alteration of the exocrine pancreas in obesity has not been clarified. In the present study, we investigated biochemical and histological changes in the exocrine pancreas of obese model rats. Methods Zucker lean rats were fed a standard diet, and Zucker diabetic fatty (ZDF) rats were divided into 2 groups fed a standard diet and a high-fat diet, respectively. These experimental groups were fed each of the diets from 6 weeks until 12, 18, 24 weeks of age. We performed blood biochemical assays and histological analysis of the pancreas. Results In the ZDF rats fed a high-fat diet, the ratio of accumulated pancreatic fat area relative to exocrine gland area was increased significantly at 18 weeks of age in comparison with the other 2 groups (P < 0.05), and lipid droplets were observed in acinar cells. Subsequently, at 24 weeks of age in this group, pancreatic fibrosis and the serum exocrine pancreatic enzyme levels were increased significantly relative to the other 2 groups (P < 0.01). Conclusions In ZDF rats fed a chronic high-fat diet, fat accumulates in pancreatic acinar cells, and this fatty change seems to be related to subsequent pancreatic fibrosis and acinar cell injury. PMID:24717823

Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

PubMed

Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

2009-01-01

To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

Co-occurrence of tannin and tannin-less vacuoles in sensitive plants.

PubMed

Fleurat-Lessard, Pierrette; Béré, Emile; Lallemand, Magali; Dédaldéchamp, Fabienne; Roblin, Gabriel

2016-05-01

Vacuoles of different types frequently coexist in the same plant cell, but the duality of the tannin/tannin-less vacuoles observed in Mimosa pudica L. is rare. In this plant, which is characterized by highly motile leaves, the development and original features of the double vacuolar compartment were detailed in primary pulvini from the young to the mature leaf stage. In young pulvini, the differentiation of tannin vacuoles first occurred in the epidermis and progressively spread toward the inner cortex. In motor cells of nonmotile pulvini, tannin deposits first lined the membranes of small vacuole profiles and then formed opaque clusters that joined together to form a large tannin vacuole (TV), the proportion of which in the cell was approximately 45%. At this stage, transparent vacuole profiles were rare and small, but as the parenchyma cells enlarged, these profiles coalesced to form a transparent vacuole with a convexity toward the larger-sized tannin vacuole. When leaf motility began to occur, the two vacuole types reached the same relative proportion (approximately 30%). Finally, in mature cells displaying maximum motility, the large transparent colloidal vacuole (CV) showed a relative proportion increasing to approximately 50%. At this stage, the proportion of the tannin vacuole, occurring in the vicinity of the nucleus, decreased to approximately 10%. The presence of the condensed type of tannins (proanthocyanidins) was proven by detecting their fluorescence under UV light and by specific chemical staining. This dual vacuolar profile was also observed in nonmotile parts of M. pudica (e.g., the petiole and the stem). Additional observations of leaflet pulvini showing more or less rapid movements showed that this double vacuolar structure was present in certain plants (Mimosa spegazzinii and Desmodium gyrans), but absent in others (Albizzia julibrissin, Biophytum sensitivum, and Cassia fasciculata). Taken together, these observations strongly suggest that a

The exocrine pancreas: the acinar-ductal tango in physiology and pathophysiology.

PubMed

Hegyi, Peter; Petersen, Ole H

2013-01-01

There are many reviews of pancreatic acinar cell function and also of pancreatic duct function, but there is an almost total absence of synthetic reviews bringing the integrated functions of these two vitally and mutually interdependent cells together. This is what we have attempted to do in this chapter. In the first part, we review the normal integrated function of the acinar-ductal system, with particular emphasis on how regulation of one type of cell also influences the other cell type. In the second part, we review a range of pathological processes, particularly those involved in acute pancreatitis (AP), an often-fatal human disease in which the pancreas digests itself, in order to explore how malfunction of one of the cell types adversely affects the function of the other.

Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

PubMed

Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

2014-09-05

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Wortmannin-induced vacuole fusion enhances amyloplast dynamics in Arabidopsis zigzag1 hypocotyls

PubMed Central

Alvarez, Ashley Ann; Han, Sang Won; Toyota, Masatsugu; Brillada, Carla; Zheng, Jiameng; Gilroy, Simon

2016-01-01

Gravitropism in Arabidopsis shoots depends on the sedimentation of amyloplasts in the endodermis, and a complex interplay between the vacuole and F-actin. Gravity response is inhibited in zigzag-1 (zig-1), a mutant allele of VTI11, which encodes a SNARE protein involved in vacuole fusion. zig-1 seedlings have fragmented vacuoles that fuse after treatment with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and underscore a role of phosphoinositides in vacuole fusion. Using live-cell imaging with a vertical stage microscope, we determined that young endodermal cells below the apical hook that are smaller than 70 μm in length are the graviperceptive cells in dark-grown hypocotyls. This result was confirmed by local wortmannin application to the top of zig-1 hypocotyls, which enhanced shoot gravitropism in zig-1 mutants. Live-cell imaging of zig-1 hypocotyl endodermal cells indicated that amyloplasts are trapped between juxtaposed vacuoles and their movement is severely restricted. Wortmannin-induced fusion of vacuoles in zig-1 seedlings increased the formation of transvacuolar strands, enhanced amyloplast sedimentation and partially suppressed the agravitropic phenotype of zig-1 seedlings. Hypergravity conditions at 10 g were not sufficient to displace amyloplasts in zig-1, suggesting the existence of a physical tether between the vacuole and amyloplasts. Our results overall suggest that vacuole membrane remodeling may be involved in regulating the association of vacuoles and amyloplasts during graviperception. PMID:27816929

Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.

PubMed

Gao, Xiao-Yu; Liao, Xing-Cheng; Wu, Ruo-Lai; Liu, Ting; Wang, Hai-Xing; Lu, Ling-Li

This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.

Transdifferentiation of mouse adipose-derived stromal cells into acinar cells of the submandibular gland using a co-culture system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jingu; Park, Sangkyu; Roh, Sangho, E-mail: sangho@snu.ac.kr

A loss of salivary gland function often occurs after radiation therapy in head and neck tumors, though secretion of saliva by the salivary glands is essential for the health and maintenance of the oral environment. Transplantation of salivary acinar cells (ACs), in part, may overcome the side effects of therapy. Here we directly differentiated mouse adipose-derived stromal cells (ADSCs) into ACs using a co-culture system. Multipotent ADSCs can be easily collected from stromal vascular fractions of adipose tissues. The isolated ADSCs showed positive expression of markers such as integrin beta-1 (CD29), cell surface glycoprotein (CD44), endoglin (CD105), and Nanog. Themore » cells were able to differentiate into adipocytes, osteoblasts, and neural-like cells after 14 days in culture. ADSCs at passage 2 were co-cultured with mouse ACs in AC culture medium using the double-chamber (co-culture system) to avoid mixing the cell types. The ADSCs in this co-culture system expressed markers of ACs, such as α-amylases and aquaporin5, in both mRNA and protein. ADSCs cultured in AC-conditioned medium also expressed AC markers. Cellular proliferation and senescence analyses demonstrated that cells in the co-culture group showed lower senescence and a higher proliferation rate than the AC-conditioned medium group at Days 14 and 21. The results above imply direct conversion of ADSCs into ACs under the co-culture system; therefore, ADSCs may be a stem cell source for the therapy for salivary gland damage. - Highlights: • ADSCs could transdifferentiate into acinar cells (ACs) using ACs co-culture (CCA). • Transdifferentiated ADSCs expressed ACs markers such as α-amylase and aquaporin5. • High proliferation and low senescence were presented in CCA at Day 14. • Transdifferentiation of ADSCs into ACs using CCA may be an appropriate method for cell-based therapy.« less

Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

PubMed

Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

2016-05-09

It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model. Copyright © 2016 Elsevier Ltd. All rights reserved.

Escape of Actively Secreting Shigella flexneri from ATG8/LC3-Positive Vacuoles Formed during Cell-To-Cell Spread Is Facilitated by IcsB and VirA

PubMed Central

Sachse, Martin; Sansonetti, Philippe J.; Parsot, Claude

2015-01-01

ABSTRACT The enteropathogenic bacterium Shigella flexneri uses a type 3 secretion apparatus (T3SA) to transfer proteins dubbed translocators and effectors inside host cells, inducing bacterial uptake and subsequent lysis of the entry vacuole. Once in the cytoplasm, the outer membrane protein IcsA induces actin polymerization, enabling cytoplasmic movement and cell-to-cell spread of bacteria. During this infectious process, S. flexneri is targeted by ATG8/LC3. The effector IcsB was proposed to inhibit LC3 recruitment by masking a region of IcsA recognized by the autophagy pathway component ATG5. The effector VirA, a GTPase-activating protein (GAP) for Rab1, was also shown to prevent LC3 recruitment. However, the context of LC3 recruitment around S. flexneri is not fully understood. Here, we show that LC3 is recruited specifically around secreting bacteria that are still present in vacuoles formed during entry and cell-to-cell spread. While LC3 recruitment occurs around a small proportion of intracellular wild-type bacteria, the icsB, virA, and icsB virA mutants display incremental defaults in escape from LC3-positive vacuoles formed during cell-to-cell spread. Our results indicate that IcsB and VirA act synergistically to allow bacteria to escape from LC3-positive vacuoles by acting at or in the immediate vicinity of the vacuole membrane(s). We also demonstrate that LC3 is recruited around bacteria still present in the single-membrane entry vacuole, in a manner akin to that seen with LC3-associated phagocytosis. Our results indicate that LC3 recruitment occurs around bacteria still, or already, in membrane compartments formed during entry and cell-to-cell spread, and not around bacteria free in the cytoplasm. PMID:26015503

Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar-to-ductal metaplasia.

PubMed

Bombardo, Marta; Saponara, Enrica; Malagola, Ermanno; Chen, Rong; Seleznik, Gitta M; Haumaitre, Cecile; Quilichini, Evans; Zabel, Anja; Reding, Theresia; Graf, Rolf; Sonda, Sabrina

2017-11-01

Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease. We analysed HDAC regulation during cerulein-induced acute, chronic and autoimmune pancreatitis using different transgenic mouse models. The functional relevance of class I HDACs was tested with the selective inhibitor MS-275 in vivo upon pancreatitis induction and in vitro in activated macrophages and primary acinar cell explants. HDAC expression and activity were up-regulated in a time-dependent manner following induction of pancreatitis, with the highest abundance observed for class I HDACs. Class I HDAC inhibition did not prevent the initial acinar cell damage. However, it effectively reduced the infiltration of inflammatory cells, including macrophages and T cells, in both acute and chronic phases of the disease, and directly disrupted macrophage activation. In addition, MS-275 treatment reduced DNA damage in acinar cells and limited acinar de-differentiation into acinar-to-ductal metaplasia in a cell-autonomous manner by impeding the EGF receptor signalling axis. These results demonstrate that class I HDACs are critically involved in the development of acute and chronic forms of pancreatitis and suggest that blockade of class I HDAC isoforms is a promising target to improve the outcome of the disease. © 2017 The British Pharmacological Society.

Ethanol exerts dual effects on calcium homeostasis in CCK-8-stimulated mouse pancreatic acinar cells.

PubMed

Fernández-Sánchez, Marcela; del Castillo-Vaquero, Angel; Salido, Ginés M; González, Antonio

2009-10-30

A significant percentage of patients with pancreatitis often presents a history of excessive alcohol consumption. Nevertheless, the patho-physiological effect of ethanol on pancreatitis remains poorly understood. In the present study, we have investigated the early effects of acute ethanol exposure on CCK-8-evoked Ca2+ signals in mouse pancreatic acinar cells. Changes in [Ca2+]i and ROS production were analyzed employing fluorescence techniques after loading cells with fura-2 or CM-H2DCFDA, respectively. Ethanol, in the concentration range from 1 to 50 mM, evoked an oscillatory pattern in [Ca2+]i. In addition, ethanol evoked reactive oxygen species generation (ROS) production. Stimulation of cells with 1 nM or 20 pM CCK-8, respectively led to a transient change and oscillations in [Ca2+]i. In the presence of ethanol a transformation of 20 pM CCK-8-evoked physiological oscillations into a single transient increase in [Ca2+]i in the majority of cells was observed. Whereas, in response to 1 nM CCK-8, the total Ca2+ mobilization was significantly increased by ethanol pre-treatment. Preincubation of cells with 1 mM 4-MP, an inhibitor of alcohol dehydrogenase, or 10 microM of the antioxidant cinnamtannin B-1, reverted the effect of ethanol on total Ca2+ mobilization evoked by 1 nM CCK-8. Cinnamtannin B-1 blocked ethanol-evoked ROS production. ethanol may lead, either directly or through ROS generation, to an over stimulation of pancreatic acinar cells in response to CCK-8, resulting in a higher Ca2+ mobilization compared to normal conditions. The actions of ethanol on CCK-8-stimulation of cells create a situation potentially leading to Ca2+ overload, which is a common pathological precursor that mediates pancreatitis.

Beneficial effect of the bioflavonoid quercetin on cholecystokinin-induced mitochondrial dysfunction in isolated rat pancreatic acinar cells.

PubMed

Weber, Heike; Jonas, Ludwig; Wakileh, Michael; Krüger, Burkhard

2014-03-01

The pathogenesis of acute pancreatitis (AP) is still poorly understood. Thus, a reliable pharmacological therapy is currently lacking. In recent years, an impairment of the energy metabolism of pancreatic acinar cells, caused by Ca(2+)-mediated depolarization of the inner mitochondrial membrane and a decreased ATP supply, has been implicated as an important pathological event. In this study, we investigated whether quercetin exerts protection against mitochondrial dysfunction. Following treatment with or without quercetin, rat pancreatic acinar cells were stimulated with supramaximal cholecystokinin-8 (CCK). CCK caused a decrease in the mitochondrial membrane potential (MMP) and ATP concentration, whereas the mitochondrial dehydrogenase activity was significantly increased. Quercetin treatment before CCK application exerted no protection on MMP but increased ATP to a normal level, leading to a continuous decrease in the dehydrogenase activity. The protective effect of quercetin on mitochondrial function was accompanied by a reduction in CCK-induced changes to the cell membrane. Concerning the molecular mechanism underlying the protective effect of quercetin, an increased AMP/ATP ratio suggests that the AMP-activated protein kinase system may be activated. In addition, quercetin strongly inhibited CCK-induced trypsin activity. The results indicate that the use of quercetin may be a therapeutic strategy for reducing the severity of AP.

Pancreatic acinar cells: ionic dependence of acetylcholine-induced membrane potential and resistance change.

PubMed Central

Nishiyama, A; Petersen, O H

1975-01-01

1. Intracellular recordings of membrane potential, input resistance and time constant have been made in vitro from the exocrine acinar cells of the mouse pancreas using glass micro-electrodes. The acinar cells were stimulated by acetylcholine (ACh). In some cases ACh was simply directly added to the tissue superfusion bath, in other experiments ACh was applied locally to pancreatic acini by micro-iontophoresis. 2. Current-voltage relations were investigated by injecting rectangular de- or hyperpolarizing current pulses through the recording micro-electrode. Within a relatively wide range (-20 to -70 mV) there was a linear relation between injected current and change in membrane potential. The slope of such linear curves corresponded to an input resistance of about 3-8 M omega. The membrane time constant was about 5-10 msec. 3. ACh depolarized the cell membrane and caused a marked reduction of input resistance and time constant. The minimum latency of the ACh-induced depolarization (microiontophoretic application) was 100-300 msec. Maximal depolarization was about 20 mV. The effect of this local ACh application was abolished by atropine (1-4 x 10-6 M). The blocking effect of atropine was fully reversible. 4. Stimulating with ACh during the passage of large depolarizing current pulses made it possible simultaneously to observe the effect of ACh at two different levels of resting potential (RP). At the spontaneous RP of about minus 40 mV ACh evoked a depolarization of usual magnitude (15-20 mV) while at the artificially displaced level of about -10 mV a small hyperpolarization (about 5 mV) was observed. It therefore appears that the reversal potential of the transmitter equilibrium potential is about -20 mV. 5. Replacement of the superfusion fluid C1 by sulphate or methylsulphate caused an initial short-lasting depolarization, thereafter the normal resting potential was reassumed... PMID:1142124

Prostate specific antigen and acinar density: a new dimension, the "Prostatocrit".

PubMed

Robinson, Simon; Laniado, Marc; Montgomery, Bruce

2017-01-01

Prostate-specific antigen densities have limited success in diagnosing prostate cancer. We emphasise the importance of the peripheral zone when considered with its cellular constituents, the "prostatocrit". Using zonal volumes and asymmetry of glandular acini, we generate a peripheral zone acinar volume and density. With the ratio to the whole gland, we can better predict high grade and all grade cancer. We can model the gland into its acinar and stromal elements. This new "prostatocrit" model could offer more accurate nomograms for biopsy. 674 patients underwent TRUS and biopsy. Whole gland and zonal volumes were recorded. We compared ratio and acinar volumes when added to a "clinic" model using traditional PSA density. Univariate logistic regression was used to find significant predictors for all and high grade cancer. Backwards multiple logistic regression was used to generate ROC curves comparing the new model to conventional density and PSA alone. Prediction of all grades of prostate cancer: significant variables revealed four significant "prostatocrit" parameters: log peripheral zone acinar density; peripheral zone acinar volume/whole gland acinar volume; peripheral zone acinar density/whole gland volume; peripheral zone acinar density. Acinar model (AUC 0.774), clinic model (AUC 0.745) (P=0.0105). Prediction of high grade prostate cancer: peripheral zone acinar density ("prostatocrit") was the only significant density predictor. Acinar model (AUC 0.811), clinic model (AUC 0.769) (P=0.0005). There is renewed use for ratio and "prostatocrit" density of the peripheral zone in predicting cancer. This outperforms all traditional density measurements. Copyright® by the International Brazilian Journal of Urology.

Glial S100B Positive Vacuoles In Purkinje Cells: Earliest Morphological Abnormality In SCA1 Transgenic Mice

PubMed Central

VIG, Parminder J.S.; LOPEZ, Maripar E.; WEI, Jinrong; D’SOUZA, David R.; SUBRAMONY, SH; HENEGAR, Jeffrey; FRATKIN, Jonathan D.

2007-01-01

Spinocerebellar ataxia-1 (SCA1) is caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-1. The overexpression of mutant ataxin-1 in SCA1 transgenic mice results in the formation of cytoplasmic vacuoles in Purkinje neurons (PKN) of the cerebellum. PKN are closely associated with neighboring Bergmann glia. To elucidate the role of Bergmann glia in SCA1 pathogenesis, cerebellar tissue from 7 days to 6 wks old SCA1 transgenic and wildtype mice were used. We observed that Bergmann glial S100B protein is localized to the cytoplasmic vacuoles in SCA1 PKN. These S100B positive cytoplasmic vacuoles began appearing much before the onset of behavioral abnormalities, and were negative for other glial and PKN marker proteins. Electron micrographs revealed that vacuoles have a double membrane. In the vacuoles, S100B colocalized with receptors of advanced glycation end-products (RAGE), and S100B co-immunoprecipated with cerebellar RAGE. In SCA1 PKN cultures, exogenous S100B protein interacted with the PKN membranes and was internalized. These data suggest that glial S100B though extrinsic to PKN is sequestered into cytoplasmic vacuoles in SCA1 mice at early postnatal ages. Further, S100B may be binding to RAGE on Purkinje cell membranes before these membranes are internalized. PMID:18176630

Isoproterenol-stimulated labelling of particulate proteins by using [adenylate-32P]NAD+ independent on a cAMP-dependent protein kinase in parotid acinar cells.

PubMed

Sugiya, H; Hara-Yokoyama, M; Furuyama, S

1992-03-30

When saponin-permeabilized rat parotid acinar cells were incubated with [adenylate-32P]NAD+, labelling of proteins (33, 27 and 23 kDa) in particulate fractions of the cells was stimulated by isoproterenol. The effect of isoproterenol was completely blocked by a beta-antagonist. Both forskolin or cAMP mimicked the effect of isoproterenol on the labelling. However, an inhibitor of cAMPdPK failed to induce complete inhibition of the effects of isoproterenol, forskolin and cAMP. When the labelled proteins were treated with snake venom phosphodiesterase, neither [32P]5'-AMP nor [32P]phosphoribosyladenosine was released. These results suggest that covalent modification of proteins with NAD+, which is distinct from ADP-ribosylation and cAMPdPK-dependent phosphorylation, is coupled to beta-receptor-cAMP signalling system in rat parotid acinar cells.

Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

PubMed

Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

2015-11-01

Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

PubMed

Li, Dong Xiao; Hu, Hai Yan; Li, Gan; Ru, Zhen Gang; Tian, Hui Qiao

2017-09-01

Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca 2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

Role of alveolar topology on acinar flows and convective mixing.

PubMed

Hofemeier, Philipp; Sznitman, Josué

2014-06-01

Due to experimental challenges, computational simulations are often sought to quantify inhaled aerosol transport in the pulmonary acinus. Commonly, these are performed using generic alveolar topologies, including spheres, toroids, and polyhedra, to mimic the complex acinar morphology. Yet, local acinar flows and ensuing particle transport are anticipated to be influenced by the specific morphological structures. We have assessed a range of acinar models under self-similar breathing conditions with respect to alveolar flow patterns, convective flow mixing, and deposition of fine particles (1.3 μm diameter). By tracking passive tracers over cumulative breathing cycles, we find that irreversible flow mixing correlates with the location and strength of the recirculating vortex inside the cavity. Such effects are strongest in proximal acinar generations where the ratio of alveolar to ductal flow rates is low and interalveolar disparities are most apparent. Our results for multi-alveolated acinar ducts highlight that fine 1 μm inhaled particles subject to alveolar flows are sensitive to the alveolar topology, underlining interalveolar disparities in particle deposition patterns. Despite the simplicity of the acinar models investigated, our findings suggest that alveolar topologies influence more significantly local flow patterns and deposition sites of fine particles for upper generations emphasizing the importance of the selected acinar model. In distal acinar generations, however, the alveolar geometry primarily needs to mimic the space-filling alveolar arrangement dictated by lung morphology.

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

PubMed

Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

2015-04-01

The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

Multinodular and Vacuolating Neuronal Tumor: A Rare Seizure-associated Entity.

PubMed

Cathcart, Sahara J; Klug, Jeffrey R; Helvey, Jason T; L White, Matthew; Gard, Andrew P; McComb, Rodney D

2017-07-01

Multinodular and vacuolating neuronal tumor is a recently described seizure-associated entity with overlapping features of a malformative and neoplastic process. We report a case of multinodular and vacuolating neuronal tumor in a 29-year-old man with a history of recent headaches and complex partial seizures. Neuroimaging revealed a nonenhancing, T2 and T2 fluid-attenuated inversion recovery hyperintense multinodular lesion in the right temporal lobe. Lesional tissue demonstrated well-demarcated nodules of ganglioid cells with vacuolation of both the perikarya and the fibrillary neuropil-like background. The ganglioid cells showed weak cytoplasmic reactivity for synaptophysin and were nonreactive for neurofilament and chromogranin. CD34-positive stellate cells were present within the nodules. A 50-gene next-generation sequencing panel did not identify any somatic mutations in genomic DNA extracted from the tumor.

Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis

PubMed Central

Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda

2016-01-01

The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol. PMID:26975994

Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

PubMed Central

Park, Miyoung; Martins, Vicente P.; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

2011-01-01

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism

Megamitochondria in the serous acinar cells of the submandibular gland of the neotropical fruit bat, Artibeus obscurus.

PubMed

Tandler, B; Nagato, T; Phillips, C J

1997-05-01

As part of a continuing investigation of the comparative ultrastructure of chiropteran salivary glands, we examined the submandibular glands of eight species of neotropical fruit bats in the genus Artibeus. We previously described secretory granules of unusual substructure in the seromucous demilunar cells of this organ in some species in this genus. In the present study, we turned our attention to the serous acinar cells in the same glands. Specimens of eight species of Artibeus were collected in neotropical localities. Salivary glands were extirpated in the field and thin slices were fixed by immersion in triple aldehyde-DMSO or in modified half-strength Karnovsky's fixative. Tissues were further processed for electron microscopy by conventional means. In contrast to seromucous cells, which exhibit species-specific diversification in bats of this genus, the secretory apparatus and secretory granules in the serous acinar cells are highly conserved across all seven species. The single exception involves the mitochondria in one species. In this instance, some of the serous cell mitochondria in Artibeus obscurus are modified into megamitochondria. Such organelles usually have short, peripheral cristae; a laminar inclusion is present in the matrix compartment of every outsized organelle. Inclusions of this nature never are present in normal-size mitochondria in the serous cells. None of the megamitochondria were observed in the process of degeneration. The giant mitochondria in A. obscurus have a matrical structure that is radically different from that of the only other megamitochondria reported to occur in bat salivary glands. The factors that lead to variation in megamitochondrial substructure in different species, as well as the functional capacities of such giant organelles, are unknown.

The role of anisotropic expansion for pulmonary acinar aerosol deposition

PubMed Central

Hofemeier, Philipp; Sznitman, Josué

2016-01-01

Lung deformations at the local pulmonary acinar scale are intrinsically anisotropic. Despite progress in imaging modalities, the true heterogeneous nature of acinar expansion during breathing remains controversial, where our understanding of inhaled aerosol deposition still widely emanates from studies under self-similar, isotropic wall motions. Building on recent 3D models of multi-generation acinar networks, we explore in numerical simulations how different hypothesized scenarios of anisotropic expansion influence deposition outcomes of inhaled aerosols in the acinar depths. While the broader range of particles acknowledged to reach the acinar region (dp = 0.005–5.0 μm) are largely unaffected by the details of anisotropic expansion under tidal breathing, our results suggest nevertheless that anisotropy modulates the deposition sites and fractions for a narrow band of sub-micron particles (dp ~ 0.5–0.75 μm), where the fate of aerosols is greatly intertwined with local convective flows. Our findings underscore how intrinsic aerosol motion (i.e. diffusion, sedimentation) undermines the role of anisotropic wall expansion that is often attributed in determining aerosol mixing and acinar deposition. PMID:27614613

Dietary-induced changes in the fatty acid profile of rat pancreatic membranes are associated with modifications in acinar cell function and signalling.

PubMed

Yago, Maria D; Diaz, Ricardo J; Ramirez, Rolando; Martinez, Maria A; Mañas, Mariano; Martinez-Victoria, Emilio

2004-02-01

The effects of dietary lipids on the fatty acid composition of rat pancreatic membranes and acinar cell function were investigated. Weaning rats were fed for 8 weeks on one of two diets which contained 100 g virgin olive oil (OO) or sunflower-seed oil (SO)/kg. Pancreatic plasma membranes were isolated and fatty acids determined. Amylase secretion and cytosolic concentrations of Ca(2+) and Mg(2+) were measured in pancreatic acini. Membrane fatty acids were profoundly affected by the diets; the rats fed OO had higher levels of 18 : 1n-9 (42.86 (sem 1.99) %) and total MUFA compared with the animals fed SO (25.37 (sem 1.11) %). Reciprocally, the SO diet resulted in greater levels of total and n-6 PUFA than the OO diet. The most striking effect was observed for 18 : 2n-6 (SO 17.88 (sem 1.32) %; OO 4.45 (sem 0.60) %), although the levels of 20 : 4n-6 were also different. The proportion of total saturated fatty acids was similar in both groups, and there was only a slight, not significant (P=0.098), effect on the unsaturation index. Compared with the OO group, acinar cells from the rats fed SO secreted more amylase at rest but less in response to cholecystokinin octapeptide, and this was paralleled by reduced Ca(2+) responses to the secretagogue. The results confirm that rat pancreatic cell membranes are strongly influenced by the type of dietary fat consumed and this is accompanied by a modulation of the secretory activity of pancreatic acinar cells that involves, at least in part, Ca(2+) signalling.

Pumping up the volume - vacuole biogenesis in Arabidopsis thaliana.

PubMed

Krüger, Falco; Schumacher, Karin

2017-07-08

Plant architecture follows the need to collect CO 2, solar energy, water and mineral nutrients via large surface areas. It is by the presence of a central vacuole that fills much of the cell volume that plants manage to grow at low metabolic cost. In addition vacuoles buffer the fluctuating supply of essential nutrients and help to detoxify the cytosol when plants are challenged by harmful molecules. Despite their large size and multiple important functions, our knowledge of vacuole biogenesis and the machinery underlying their amazing dynamics is still fragmentary. In this review, we try to reconcile past and present models for vacuole biogenesis with the current knowledge of multiple parallel vacuolar trafficking pathways and the molecular machineries driving membrane fusion and organelle shape. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dexamethasone treatment induces the reprogramming of pancreatic acinar cells to hepatocytes and ductal cells.

PubMed

Al-Adsani, Amani; Burke, Zoë D; Eberhard, Daniel; Lawrence, Katherine L; Shen, Chia-Ning; Rustgi, Anil K; Sakaue, Hiroshi; Farrant, J Mark; Tosh, David

2010-10-27

The pancreatic exocrine cell line AR42J-B13 can be reprogrammed to hepatocytes following treatment with dexamethasone. The question arises whether dexamethasone also has the capacity to induce ductal cells as well as hepatocytes. AR42J-B13 cells were treated with and without dexamethasone and analyzed for the expression of pancreatic exocrine, hepatocyte and ductal markers. Addition of dexamethasone inhibited pancreatic amylase expression, induced expression of the hepatocyte marker transferrin as well as markers typical of ductal cells: cytokeratin 7 and 19 and the lectin peanut agglutinin. However, the number of ductal cells was low compared to hepatocytes. The proportion of ductal cells was enhanced by culture with dexamethasone and epidermal growth factor (EGF). We established several features of the mechanism underlying the transdifferentiation of pancreatic exocrine cells to ductal cells. Using a CK19 promoter reporter, we show that a proportion of the ductal cells arise from differentiated pancreatic exocrine-like cells. We also examined whether C/EBPβ (a transcription factor important in the conversion of pancreatic cells to hepatocytes) could alter the conversion from acinar cells to a ductal phenotype. Overexpression of an activated form of C/EBPβ in dexamethasone/EGF-treated cells provoked the expression of hepatocyte markers and inhibited the expression of ductal markers. Conversely, ectopic expression of a dominant-negative form of C/EBPβ, liver inhibitory protein, inhibited hepatocyte formation in dexamethasone-treated cultures and enhanced the ductal phenotype. These results indicate that hepatocytes and ductal cells may be induced from pancreatic exocrine AR42J-B13 cells following treatment with dexamethasone. The conversion from pancreatic to hepatocyte or ductal cells is dependent upon the expression of C/EBPβ.

IL-1 beta and IL-6 in mouse parotid acinar cells: characterization of synthesis, storage, and release.

PubMed

Tanda, N; Ohyama, H; Yamakawa, M; Ericsson, M; Tsuji, T; McBride, J; Elovic, A; Wong, D T; Login, G R

1998-01-01

Synthesis, storage, and secretion of the proinflammatory cytokine interleukin-1 beta (IL-1 beta) and the anti-inflammatory cytokine IL-6 have not been established in normal exocrine gland secretory cells. Parotid glands and isolated acinar cells prepared from BALB/c mice were homogenized for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR). IL-1 beta and IL-6 enzyme-linked immunosorbent assays (ELISAs) were done on supernatants prepared from mouse parotid acinar cell (MPAC) preparations unstimulated or stimulated between 0 and 10 min with 10(-5) M norepinephrine at 37 degrees C. MPACs were fixed in paraformaldehyde, frozen sectioned for light and electron microscopy, and labeled with antibodies to IL-1 beta and IL-6. Mouse specific riboprobes to IL-1 and IL-6 were used for in situ hybridization. RT-PCR yielded the expected IL-1 (336-bp) and IL-6 (614-bp) mRNA products. By ELISA, stimulated MPACs showed a significant increase in IL-1 beta (P < 0.03) and IL-6 (P < 0.01) release into supernatants by 10 min that paralleled the time course of amylase release. In situ hybridization showed the presence of transcripts for IL-1 and IL-6 in glandular epithelial cells. Gold-labeled IL-1 beta and IL-6 were significantly higher (P < 0.01) in granules than in the nucleus and cytoplasm. This study shows that MPACs synthesize IL-1 beta and IL-6 and release these cytokines from their granules after alpha- and beta-adrenergic stimulation.

The role of anisotropic expansion for pulmonary acinar aerosol deposition.

PubMed

Hofemeier, Philipp; Sznitman, Josué

2016-10-03

Lung deformations at the local pulmonary acinar scale are intrinsically anisotropic. Despite progress in imaging modalities, the true heterogeneous nature of acinar expansion during breathing remains controversial, where our understanding of inhaled aerosol deposition still widely emanates from studies under self-similar, isotropic wall motions. Building on recent 3D models of multi-generation acinar networks, we explore in numerical simulations how different hypothesized scenarios of anisotropic expansion influence deposition outcomes of inhaled aerosols in the acinar depths. While the broader range of particles acknowledged to reach the acinar region (d p =0.005-5.0μm) are largely unaffected by the details of anisotropic expansion under tidal breathing, our results suggest nevertheless that anisotropy modulates the deposition sites and fractions for a narrow band of sub-micron particles (d p ~0.5-0.75μm), where the fate of aerosols is greatly intertwined with local convective flows. Our findings underscore how intrinsic aerosol motion (i.e. diffusion, sedimentation) undermines the role of anisotropic wall expansion that is often attributed in determining aerosol mixing and acinar deposition. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis*

PubMed Central

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F. Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M.

2016-01-01

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. PMID:27226576

Cathepsin B Activity Initiates Apoptosis via Digestive Protease Activation in Pancreatic Acinar Cells and Experimental Pancreatitis.

PubMed

Sendler, Matthias; Maertin, Sandrina; John, Daniel; Persike, Maria; Weiss, F Ulrich; Krüger, Burkhard; Wartmann, Thomas; Wagh, Preshit; Halangk, Walter; Schaschke, Norbert; Mayerle, Julia; Lerch, Markus M

2016-07-08

Pancreatitis is associated with premature activation of digestive proteases in the pancreas. The lysosomal hydrolase cathepsin B (CTSB) is a known activator of trypsinogen, and its deletion reduces disease severity in experimental pancreatitis. Here we studied the activation mechanism and subcellular compartment in which CTSB regulates protease activation and cellular injury. Cholecystokinin (CCK) increased the activity of CTSB, cathepsin L, trypsin, chymotrypsin, and caspase 3 in vivo and in vitro and induced redistribution of CTSB to a secretory vesicle-enriched fraction. Neither CTSB protein nor activity redistributed to the cytosol, where the CTSB inhibitors cystatin-B/C were abundantly present. Deletion of CTSB reduced and deletion of cathepsin L increased intracellular trypsin activation. CTSB deletion also abolished CCK-induced caspase 3 activation, apoptosis-inducing factor, as well as X-linked inhibitor of apoptosis protein degradation, but these depended on trypsinogen activation via CTSB. Raising the vesicular pH, but not trypsin inhibition, reduced CTSB activity. Trypsin inhibition did not affect apoptosis in hepatocytes. Deletion of CTSB affected apoptotic but not necrotic acinar cell death. In summary, CTSB in pancreatitis undergoes activation in a secretory, vesicular, and acidic compartment where it activates trypsinogen. Its deletion or inhibition regulates acinar cell apoptosis but not necrosis in two models of pancreatitis. Caspase 3-mediated apoptosis depends on intravesicular trypsinogen activation induced by CTSB, not CTSB activity directly, and this mechanism is pancreas-specific. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The parasitophorous vacuole of Encephalitozoon cuniculi: biogenesis and characteristics of the host cell-pathogen interface.

PubMed

Bohne, Wolfgang; Böttcher, Karin; Gross, Uwe

2011-06-01

Microsporidia are obligate intracellular fungal pathogens of increasing importance in immunocompromised patients. They have developed a unique invasion mechanism, which is based on the explosive discharge of a hollow tubulus, the so-called polar tube. The infectious sporoplasm is subsequently extruded through this flexible tube and injected into the host cell. The model microsporidium Encephalitozoon cuniculi is a paradigm of a fungus with an extreme host cell dependency. This human pathogen possesses one of the smallest eukaryotic genomes (<3MB) identified so far and has reduced its own biosynthetic pathways to a minimum, thus depending on an efficient supply of metabolites from the host cell. E. cuniculi spends its entire intracellular life cycle inside a parasitophorous vacuole (PV), which is formed during invasion. We have provided here an overview of the biogenesis and characteristics of this important host cell-pathogen interface and suggest in this context a modified model for E. cuniculi invasion. According to the model, the host cell plasma membrane is not pierced by the polar tube, but is pushed at the contact site into the cell interior by the mechanical force of the expelled polar tube. This results in a channel-like invagination of the plasma membrane, from which finally the parasitophorous vacuole is pinched-off. Copyright © 2011 Elsevier GmbH. All rights reserved.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles.

PubMed

Vitali, Victoria; Sutka, Moira; Amodeo, Gabriela; Chara, Osvaldo; Ozu, Marcelo

2016-01-01

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient ( P f ) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, P f determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast P f . We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites ( P f / P s ), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles

PubMed Central

Vitali, Victoria; Sutka, Moira; Amodeo, Gabriela; Chara, Osvaldo; Ozu, Marcelo

2016-01-01

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements

Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy.

PubMed

Zhang, Ying; Yu, Chenguang; Huang, Guanyi; Wang, Changli; Wen, Longping

2010-09-07

Four rare earth oxides have been shown to induce autophagy. Interestingly, we often noticed plentiful vacuolization, which was not always involved in this autophagic process. In this study, we investigated three other rare-earth elements, including Yttrium (Y), Ytterbium (Yb), and Lanthanum (La). Autophagic effect could be induced by all of them but only Y(2)O(3) and Yb(2)O(3) could cause massive vacuolization. Y(2)O(3) and Yb(2)O(3) treated by sonication or centrifugation to reduce particle size were used to test vacuolization level in HeLa cell lines. The results showed that rare earth oxides-induced vacuolization is size-dependent and differs from autophagic pathway. To further clarify the characteristics of this autophagic process, we used MEF Atg-5 (autophagy associated gene 5) knockout cell line, and the result showed that the autophagic process induced by rare earth oxides is Atg-5-dependent and the observed vacuolization was independent from autophagy. Similar results could also be observed in our tests on 3-methyladenine(3-MA), a well-known autophagy inhibitor. In conclusion, for the first time, we clarified the relationship between massive vacuolization and autophagic process induced by rare earth oxides and pointed out the size effect of rare earth oxides on the formation of vacuoles, which give clues to further investigation on the mechanisms underlying their biological effects.

Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy

PubMed Central

Zhang, Ying; Yu, Chenguang; Huang, Guanyi; Wang, Changli; Wen, Longping

2010-01-01

Four rare earth oxides have been shown to induce autophagy. Interestingly, we often noticed plentiful vacuolization, which was not always involved in this autophagic process. In this study, we investigated three other rare-earth elements, including Yttrium (Y), Ytterbium (Yb), and Lanthanum (La). Autophagic effect could be induced by all of them but only Y2O3 and Yb2O3 could cause massive vacuolization. Y2O3 and Yb2O3 treated by sonication or centrifugation to reduce particle size were used to test vacuolization level in HeLa cell lines. The results showed that rare earth oxides-induced vacuolization is size-dependent and differs from autophagic pathway. To further clarify the characteristics of this autophagic process, we used MEF Atg-5 (autophagy associated gene 5) knockout cell line, and the result showed that the autophagic process induced by rare earth oxides is Atg-5-dependent and the observed vacuolization was independent from autophagy. Similar results could also be observed in our tests on 3-methyladenine(3-MA), a well-known autophagy inhibitor. In conclusion, for the first time, we clarified the relationship between massive vacuolization and autophagic process induced by rare earth oxides and pointed out the size effect of rare earth oxides on the formation of vacuoles, which give clues to further investigation on the mechanisms underlying their biological effects. PMID:20856835

Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

PubMed Central

Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

2016-01-01

The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

Traffic of Human α-Mannosidase in Plant Cells Suggests the Presence of a New Endoplasmic Reticulum-to-Vacuole Pathway without Involving the Golgi Complex1[W

PubMed Central

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2013-01-01

The transport of secretory proteins from the endoplasmic reticulum to the vacuole requires sorting signals as well as specific transport mechanisms. This work is focused on the transport in transgenic tobacco (Nicotiana tabacum) plants of a human α-mannosidase, MAN2B1, which is a lysosomal enzyme involved in the turnover of N-linked glycoproteins and can be used in enzyme replacement therapy. Although ubiquitously expressed, α-mannosidases are targeted to lysosomes or vacuoles through different mechanisms according to the organisms in which these proteins are produced. In tobacco cells, MAN2B1 reaches the vacuole even in the absence of mannose-6-phosphate receptors, which are responsible for its transport in animal cells. We report that MAN2B1 is targeted to the vacuole without passing through the Golgi complex. In addition, a vacuolar targeting signal that is recognized in plant cells is located in the MAN2B1 amino-terminal region. Indeed, when this amino-terminal domain is removed, the protein is retained in the endoplasmic reticulum. Moreover, when this domain is added to a plant-secreted protein, the resulting fusion protein is partially redirected to the vacuole. These results strongly suggest the existence in plants of a new type of vacuolar traffic that can be used by leaf cells to transport vacuolar proteins. PMID:23449646

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles

PubMed Central

Alvarez, Diego E.

2016-01-01

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207–238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207–238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. PMID:27068088

The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

PubMed

Alvarez, Diego E; Agaisse, Hervé

2016-06-01

Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

PubMed

Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

2011-06-15

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form. Copyright © 2010 Elsevier GmbH. All rights reserved.

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

PubMed

Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

2015-01-01

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

Live-cell imaging of rice cytological changes reveals the importance of host vacuole maintenance for biotrophic invasion by blast fungus, Magnaporthe oryzae.

PubMed

Mochizuki, Susumu; Minami, Eiichi; Nishizawa, Yoko

2015-12-01

The rice blast fungus Magnaporthe oryzae grows inside living host cells. Cytological analyses by live-cell imaging have revealed characteristics of the biotrophic invasion, particularly the extrainvasive hyphal membrane (EIHM) originating from the host plasma membrane and a host membrane-rich structure, biotrophic interfacial complex (BIC). Here, we observed rice subcellular changes associated with invasive hyphal growth using various transformants expressing specifically localized fluorescent proteins. The invasive hyphae did not penetrate across but were surrounded by the host vacuolar membrane together with EIHM even after branching. High-resolution imaging of BICs revealed that the host cytosol was accumulated at BIC with aggregated EIHM and a symplastic effector, Pwl2, in a punctate form. The vacuolar membrane did not aggregate in but closely surrounded the BIC. A good correlation was observed between the early collapse of vacuoles and damage of invasive hyphae in the first-invaded cell. Furthermore, a newly developed, long-term imaging method has revealed that the central vacuole gradually shrank until collapse, which was caused by the hyphal invasion occurring earlier in the neighboring cells than in the first-invaded cells. These data suggest that M. oryzae may suppress host vacuole collapse during early infection stages for successful infection. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Mixed acinar-endocrine carcinoma of the pancreas: new clinical and pathological features in a contemporary series.

PubMed

Yu, Run; Jih, Lily; Zhai, Jing; Nissen, Nicholas N; Colquhoun, Steven; Wolin, Edward; Dhall, Deepti

2013-04-01

The objective of this study was to characterize the novel clinical and pathological features of mixed acinar-endocrine carcinoma of the pancreas. This was a retrospective review of medical records and surgical pathology specimens of patients with a diagnosis of mixed acinar-endocrine carcinoma of the pancreas at Cedars-Sinai Medical Center between 2005 and 2011. Additional immunohistochemistry was performed on the specimens of some patients. Five patients were identified. The median age at presentation was 74 years (range, 59-89 years), and all patients were male. The presenting symptoms were all related to tumor mass effects. The median size of the tumor was 10 cm (range, 3.9-16 cm). Preoperative clinical diagnosis aided by fine-needle aspiration biopsy was incorrect in all 5 cases. Most tumors (3/5) exhibited predominantly endocrine differentiation without hormonal production. Only 10% to 30% of cells were truly amphicrine, whereas most were differentiated into either endocrine or acinar phenotype. The clinical behavior ranged from moderate to aggressive with postoperative survival from 2.5 months to more than 3 years. Four patients received neoadjuvant or adjuvant chemotherapy with variable responses. Mixed acinar-endocrine carcinoma of the pancreas appears to be not uncommon in men, may harbor predominantly endocrine component, is often misdiagnosed by cytology, and exhibits variable clinical behavior. Mixed acinar-endocrine carcinoma of the pancreas should be considered in older patients with sizable pancreatic mass and may warrant aggressive surgical resection and chemotherapy.

Shigella subverts the host recycling compartment to rupture its vacuole.

PubMed

Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

2014-10-08

Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

Murine pulmonary acinar mechanics during quasi-static inflation using synchrotron refraction-enhanced computed tomography.

PubMed

Sera, Toshihiro; Yokota, Hideo; Tanaka, Gaku; Uesugi, Kentaro; Yagi, Naoto; Schroter, Robert C

2013-07-15

We visualized pulmonary acini in the core regions of the mouse lung in situ using synchrotron refraction-enhanced computed tomography (CT) and evaluated their kinematics during quasi-static inflation. This CT system (with a cube voxel of 2.8 μm) allows excellent visualization of not just the conducting airways, but also the alveolar ducts and sacs, and tracking of the acinar shape and its deformation during inflation. The kinematics of individual alveoli and alveolar clusters with a group of terminal alveoli is influenced not only by the connecting alveolar duct and alveoli, but also by the neighboring structures. Acinar volume was not a linear function of lung volume. The alveolar duct diameter changed dramatically during inflation at low pressures and remained relatively constant above an airway pressure of ∼8 cmH2O during inflation. The ratio of acinar surface area to acinar volume indicates that acinar distension during low-pressure inflation differed from that during inflation over a higher pressure range; in particular, acinar deformation was accordion-like during low-pressure inflation. These results indicated that the alveoli and duct expand differently as total acinar volume increases and that the alveolar duct may expand predominantly during low-pressure inflation. Our findings suggest that acinar deformation in the core regions of the lung is complex and heterogeneous.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells.

PubMed

Fujiwara, Sachiko; Matsui, Tsubasa S; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

2018-01-01

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330-1057) of Solo binds to the C-terminal region (1451-1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells

PubMed Central

Matsui, Tsubasa S.; Ohashi, Kazumasa; Deguchi, Shinji; Mizuno, Kensaku

2018-01-01

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330–1057) of Solo binds to the C-terminal region (1451–1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization. PMID:29672603

Accumulation of mannitol in the cytoplasm and vacuole during the expansion of sepal cells associated with flower opening in Delphinium × belladonna cv. Bellamosum.

PubMed

Norikoshi, Ryo; Yamada, Kunio; Niki, Tomoko; Ichimura, Kazuo

2015-12-01

The role of mannitol differs from that of glucose, fructose and sucrose in sepal cell expansion associated with flower opening in Delphinium × belladonna. Sepals of Delphinium × belladonna are colored and much larger than the petals. To determine whether the role of mannitol in sepal growth associated with flower opening differs from those of ubiquitous metabolic sugars including glucose, fructose and sucrose, we investigated changes in cell number, subcellular concentrations of soluble carbohydrates, and osmotic potential in sepals during flower opening in Delphinium × belladonna cv. Bellamosum. The number of epidermal cells in the sepals did not increase from the stage when sepal pigmentation started, whereas the cell area increased during flower opening, indicating that petal growth during flower opening depends on cell expansion. Mannitol concentrations in the vacuole at three different stages were approximately 100 mM, which were much higher than the other carbohydrate concentrations, but they decreased slightly at open stage. In contrast, mannitol concentration in the cytoplasm was 56 mM at bud stage, but it increased to 104 mM at open stage. Glucose and fructose concentrations in the vacuole at open stage increased to 45 and 56 mM, respectively. Total osmotic potential in apoplast and symplast, which was partially due to soluble carbohydrates, was almost constant during flower opening. Therefore, mannitol may be acting constitutively as the main osmoticum in the vacuole where it may contribute to the maintenance of the osmotic balance between the cytoplasm and vacuole in open flowers. The role of mannitol differs from those of glucose, fructose, and sucrose in sepal cell expansion in Delphinium × belladonna.

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

PubMed Central

Ramnath, Raina Devi; Sun, Jia; Adhikari, Sharmila; Bhatia, Madhav

2007-01-01

Abstract Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini. PMID:18205703

Quantitative characterization of the protein contents of the exocrine pancreatic acinar cell by soft x-ray microscopy and advanced digital imaging methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Loo, Jr., Billy W.

2000-06-01

The study of the exocrine pancreatic acinar cell has been central to the development of models of many cellular processes, especially of protein transport and secretion. Traditional methods used to examine this system have provided a wealth of qualitative information from which mechanistic models have been inferred. However they have lacked the ability to make quantitative measurements, particularly of the distribution of protein in the cell, information critical for grounding of models in terms of magnitude and relative significance. This dissertation describes the development and application of new tools that were used to measure the protein content of the majormore » intracellular compartments in the acinar cell, particularly the zymogen granule. Soft x-ray microscopy permits image formation with high resolution and contrast determined by the underlying protein content of tissue rather than staining avidity. A sample preparation method compatible with x-ray microscopy was developed and its properties evaluated. Automatic computerized methods were developed to acquire, calibrate, and analyze large volumes of x-ray microscopic images of exocrine pancreatic tissue sections. Statistics were compiled on the protein density of several organelles, and on the protein density, size, and spatial distribution of tens of thousands of zymogen granules. The results of these measurements, and how they compare to predictions of different models of protein transport, are discussed.« less

Duct- and Acinar-Derived Pancreatic Ductal Adenocarcinomas Show Distinct Tumor Progression and Marker Expression.

PubMed

Ferreira, Rute M M; Sancho, Rocio; Messal, Hendrik A; Nye, Emma; Spencer-Dene, Bradley; Stone, Richard K; Stamp, Gordon; Rosewell, Ian; Quaglia, Alberto; Behrens, Axel

2017-10-24

The cell of origin of pancreatic ductal adenocarcinoma (PDAC) has been controversial. Here, we show that identical oncogenic drivers trigger PDAC originating from both ductal and acinar cells with similar histology but with distinct pathophysiology and marker expression dependent on cell of origin. Whereas acinar-derived tumors exhibited low AGR2 expression and were preceded by pancreatic intraepithelial neoplasias (PanINs), duct-derived tumors displayed high AGR2 and developed independently of a PanIN stage via non-mucinous lesions. Using orthotopic transplantation and chimera experiments, we demonstrate that PanIN-like lesions can be induced by PDAC as bystanders in adjacent healthy tissues, explaining the co-existence of mucinous and non-mucinous lesions and highlighting the need to distinguish between true precursor PanINs and PanIN-like bystander lesions. Our results suggest AGR2 as a tool to stratify PDAC according to cell of origin, highlight that not all PanIN-like lesions are precursors of PDAC, and add an alternative progression route to the current model of PDAC development. Copyright © 2017 Francis Crick Institute. Published by Elsevier Inc. All rights reserved.

Experimental evidence of age-related adaptive changes in human acinar airways

PubMed Central

Quirk, James D.; Sukstanskii, Alexander L.; Woods, Jason C.; Lutey, Barbara A.; Conradi, Mark S.; Gierada, David S.; Yusen, Roger D.; Castro, Mario

2015-01-01

The progressive decline of lung function with aging is associated with changes in lung structure at all levels, from conducting airways to acinar airways (alveolar ducts and sacs). While information on conducting airways is becoming available from computed tomography, in vivo information on the acinar airways is not conventionally available, even though acini occupy 95% of lung volume and serve as major gas exchange units of the lung. The objectives of this study are to measure morphometric parameters of lung acinar airways in living adult humans over a broad range of ages by using an innovative MRI-based technique, in vivo lung morphometry with hyperpolarized 3He gas, and to determine the influence of age-related differences in acinar airway morphometry on lung function. Pulmonary function tests and MRI with hyperpolarized 3He gas were performed on 24 healthy nonsmokers aged 19-71 years. The most significant age-related difference across this population was a 27% loss of alveolar depth, h, leading to a 46% increased acinar airway lumen radius, hence, decreased resistance to acinar air transport. Importantly, the data show a negative correlation between h and the pulmonary function measures forced expiratory volume in 1 s and forced vital capacity. In vivo lung morphometry provides unique information on age-related changes in lung microstructure and their influence on lung function. We hypothesize that the observed reduction of alveolar depth in subjects with advanced aging represents a remodeling process that might be a compensatory mechanism, without which the pulmonary functional decline due to other biological factors with advancing age would be significantly larger. PMID:26542518

Activated macrophages create lineage-specific microenvironments for pancreatic acinar- and β-cell regeneration in mice.

PubMed

Criscimanna, Angela; Coudriet, Gina M; Gittes, George K; Piganelli, Jon D; Esni, Farzad

2014-11-01

Although the cells that contribute to pancreatic regeneration have been widely studied, little is known about the mediators of this process. During tissue regeneration, infiltrating macrophages debride the site of injury and coordinate the repair response. We investigated the role of macrophages in pancreatic regeneration in mice. We used a saporin-conjugated antibody against CD11b to reduce the number of macrophages in mice following diphtheria toxin receptor-mediated cell ablation of pancreatic cells, and evaluated the effects on pancreatic regeneration. We analyzed expression patterns of infiltrating macrophages after cell-specific injury or from the pancreas of nonobese diabetic mice. We developed an in vitro culture system to study the ability of macrophages to induce cell-specific regeneration. Depletion of macrophages impaired pancreatic regeneration. Macrophage polarization, as assessed by expression of tumor necrosis factor-α, interleukin 6, interleukin 10, and CD206, depended on the type of injury. The signals provided by polarized macrophages promoted lineage-specific generation of acinar or endocrine cells. Macrophage from nonobese diabetic mice failed to provide signals necessary for β-cell generation. Macrophages produce cell type-specific signals required for pancreatic regeneration in mice. Additional study of these processes and signals might lead to new approaches for treating type 1 diabetes or pancreatitis. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion.

PubMed

Lee, Robert J; Harlow, Janice M; Limberis, Maria P; Wilson, James M; Foskett, J Kevin

2008-07-01

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3

Novel Tonoplast Transporters Identified Using a Proteomic Approach with Vacuoles Isolated from Cauliflower Buds1[W][OA

PubMed Central

Schmidt, Ulrike G.; Endler, Anne; Schelbert, Silvia; Brunner, Arco; Schnell, Magali; Neuhaus, H. Ekkehard; Marty-Mazars, Daniéle; Marty, Francis; Baginsky, Sacha; Martinoia, Enrico

2007-01-01

Young meristematic plant cells contain a large number of small vacuoles, while the largest part of the vacuome in mature cells is composed by a large central vacuole, occupying 80% to 90% of the cell volume. Thus far, only a limited number of vacuolar membrane proteins have been identified and characterized. The proteomic approach is a powerful tool to identify new vacuolar membrane proteins. To analyze vacuoles from growing tissues we isolated vacuoles from cauliflower (Brassica oleracea) buds, which are constituted by a large amount of small cells but also contain cells in expansion as well as fully expanded cells. Here we show that using purified cauliflower vacuoles and different extraction procedures such as saline, NaOH, acetone, and chloroform/methanol and analyzing the data against the Arabidopsis (Arabidopsis thaliana) database 102 cauliflower integral proteins and 214 peripheral proteins could be identified. The vacuolar pyrophosphatase was the most prominent protein. From the 102 identified proteins 45 proteins were already described. Nine of these, corresponding to 46% of peptides detected, are known vacuolar proteins. We identified 57 proteins (55.9%) containing at least one membrane spanning domain with unknown subcellular localization. A comparison of the newly identified proteins with expression profiles from in silico data revealed that most of them are highly expressed in young, developing tissues. To verify whether the newly identified proteins were indeed localized in the vacuole we constructed and expressed green fluorescence protein fusion proteins for five putative vacuolar membrane proteins exhibiting three to 11 transmembrane domains. Four of them, a putative organic cation transporter, a nodulin N21 family protein, a membrane protein of unknown function, and a senescence related membrane protein were localized in the vacuolar membrane, while a white-brown ATP-binding cassette transporter homolog was shown to reside in the plasma membrane

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

PubMed

Puchkov, Evgeny O

2010-06-01

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 microm size, stainable by a fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Signet-ring cell lymphoma of T-cell origin. An immunocytochemical and ultrastructural study relating giant vacuole formation to cytoplasmic sequestration of surface membrane.

PubMed

Grogan, T M; Richter, L C; Payne, C M; Rangel, C S

1985-09-01

In contrast to previous accounts of signet-ring lymphoma as a B-cell neoplasm, we report a case of signet-ring, large-cell lymphoma of T-cell lineage. Immunologic and ultrastructural studies were performed on a subcutaneous mass noted initially, as well as on an enlarged lymph node that developed later, in a 69-year-old man. Immunologic assessment indicated strong expression of T-helper antigen (Leu 3a + b), universal T-antigens (Leu 1, 5), and Ia. There was an absence of T-suppressor/cytotoxic antigen (Leu 2a), universal T-antigens (Leu 4, 9), and immunoglobulin light and heavy chains. Collectively, these findings indicate a mature T-cell lymphoma of T-helper type in an activated (Ia+) state. In contrast to previous reports of T-cell and Ia occurring solely as surface antigens, we demonstrated pools of cytoplasmic Leu 1, 3, 5 and Ia that displaced the nucleus. The ultrastructure of the giant cytoplasmic vacuoles was identical to the microvesicle-containing vacuoles reported in signet-ring cell lymphomas of B-cell lineage. In our case of T-cell lineage, we found substantial evidence of endocytosis by the neoplastic cells and numerous giant multivesicular bodies. The pools of cytoplasmic T and Ia antigens may result from abnormal internalization of surface T-antigens or the sequestration of T-antigen-containing Golgi-derived vesicles. Our combined immunologic and ultrastructural findings suggest that aberrant membrane recycling may be the common denominator of signet-ring formation in both B- and T-cell signet-ring lymphomas.

The role of septins in infections with vacuole-dwelling intracellular bacteria.

PubMed

Häcker, Georg

2017-07-29

Septins are a relatively little understood group of GTPases that form large assemblies in cells from all eukaryotes other than plants. Septins were first identified in cell division but have also been implicated in microbial infections. Septins often associate with cytoskeletal proteins - most often described for filamentous (F-) actin - and are considered cytoskeletal components themselves. Septins have increasingly been found to partake in processes that are linked to intracellular membranes, from mitochondria to phagosomes, and evidence is accumulating that septins specifically bind to membranes. Since a number of microorganisms have specialized to live and grow inside membranous vacuoles in the cytosol of mammalian cells, this membrane-association of septins suggests that septins may also be involved in the membranous, vacuolar structures that develop around these microbes. However, data are limited on this issue: septins have been identified by proteome analysis on some microbe-bearing vacuoles, but more extensive experimental data are only available for infections with the obligate intracellular bacterium Chlamydia trachomatis. In this review article I will discuss the available data and speculate about the mechanisms of recruitment and potential functions of septins for vacuole-dwelling microorganisms, which may be peculiar to Chlamydia or may pertain more generally to this class of microbes. Copyright © 2017 Elsevier GmbH. All rights reserved.

Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress

PubMed Central

Lee, Wei-Jiunn; Chien, Ming-Hsien; Chow, Jyh-Ming; Chang, Junn-Liang; Wen, Yu-Ching; Lin, Yung-Wei; Cheng, Chao-Wen; Lai, Gi-Ming; Hsiao, Michael; Lee, Liang-Ming

2015-01-01

The antiapoptotic and antiautophagic abilities of cancer cells constitute a major challenge for anticancer drug treatment. Strategies for triggering nonapoptotic or nonautophagic cell death may improve therapeutic efficacy against cancer. Curcumin has been reported to exhibit cancer chemopreventive properties. Herein, we report that curcumin induced apoptosis in LNCaP, DU145, and PC-3 cells but triggered extensive cytoplasmic vacuolation in PC-3M cells. Electron microscopic images showed that the vacuoles lacked intracellular organelles and were derived from the endoplasmic reticulum (ER). Moreover, curcumin-induced vacuolation was not reversed by an apoptosis- or autophagy-related inhibitor, suggesting that vacuolation-mediated cell death differs from classical apoptotic and autophagic cell death. Mechanistic investigations revealed that curcumin treatment upregulated the ER stress markers CHOP and Bip/GRP78 and the autophagic marker LC3-II. In addition, curcumin induced ER stress by triggering ROS generation, which was supported by the finding that treating cells with the antioxidant NAC alleviated curcumin-mediated ER stress and vacuolation-mediated death. An in vivo PC-3M orthotopic prostate cancer model revealed that curcumin reduced tumor growth by inducing ROS production followed by vacuolation-mediated cell death. Overall, our results indicated that curcumin acts as an inducer of ROS production, which leads to nonapoptotic and nonautophagic cell death via increased ER stress. PMID:26013662

Involvement of the Vacuoles of the Endodermis in the Early Process of Shoot Gravitropism in Arabidopsis

PubMed Central

Morita, Miyo Terao; Kato, Takehide; Nagafusa, Kiyoshi; Saito, Chieko; Ueda, Takashi; Nakano, Akihiko; Tasaka, Masao

2002-01-01

The endodermal cells of the shoot are thought to be the gravity-sensing cells in Arabidopsis. The amyloplasts in the endodermis that sediment in the direction of gravity may act as statoliths. Endodermis-specific expression of SGR2 and ZIG using the SCR promoter could complement the abnormal shoot gravitropism of the sgr2 and zig mutants, respectively. The abnormalities in amyloplast sedimentation observed in both mutants recovered simultaneously. These results indicate that both genes in the endodermal cell layer are crucial for shoot gravitropism. ZIG encodes AtVTI11, which is a SNARE involved in vesicle transport to the vacuole. The fusion protein of SGR2 and green fluorescent protein localized to the vacuole and small organelles. These observations indicate that ZIG and SGR2 are involved in the formation and function of the vacuole, a notion supported by the results of subcellular analysis of the sgr2 and zig mutants with electron microscopy. These results strongly suggest that the vacuole participates in the early events of gravitropism and that SGR2 and ZIG functions are involved. PMID:11826298

Effects of the type of dietary fat on acetylcholine-evoked amylase secretion and calcium mobilization in isolated rat pancreatic acinar cells.

PubMed

Yago, María D; Díaz, Ricardo J; Martínez, María A; Audi, Nama'a; Naranjo, José A; Martínez-Victoria, Emilio; Mañas, Mariano

2006-04-01

Olive oil is a major component of the Mediterranean diet, and its role in human health is being actively debated. This study aimed to clarify the mechanism of pancreatic adaptation to dietary fat. For this purpose, we examined whether dietary-induced modification of pancreatic membranes affects acinar cell function in response to the secretagogue acetylcholine (ACh). Weaning male Wistar rats were assigned to one of two experimental groups and fed for 8 weeks with a commercial chow (C) or a semisynthetic diet containing virgin olive oil as dietary fat (OO). The fatty acid composition of pancreatic plasma membranes was determined by gas-liquid chromatography. For assessment of secretory function, viable acini were incubated with ACh and amylase of supernatant was further assayed with a substrate reagent. Changes in cytosolic Ca(2+) concentration in response to ACh were measured by fura-2 AM fluorimetry. Compared to C rats, pancreatic cell membranes of OO rats had a higher level of monounsaturated fatty acids and a lower level of both saturated and polyunsaturated fatty acids, thus, reflecting the type of dietary fat given. Net amylase secretion in response to ACh was greatly enhanced after OO feeding, although this was not paralleled by enhancement of ACh-evoked Ca(2+) peak increases. In conclusion, chronic intake of diets that differ in the fat type influences not only the fatty acid composition of rat pancreatic membranes but also the responsiveness of acinar cells to ACh. This mechanism may be, at least in part, responsible for the adaptation of the exocrine pancreas to the type of fat available.

HCO3− Secretion by Murine Nasal Submucosal Gland Serous Acinar Cells during Ca2+-stimulated Fluid Secretion

PubMed Central

Lee, Robert J.; Harlow, Janice M.; Limberis, Maria P.; Wilson, James M.; Foskett, J. Kevin

2008-01-01

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca2+-activated Cl− secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca2+-activated Cl− secretion was accompanied by secretion of HCO3−, possibly a critical ASL component, by simultaneous measurements of intracellular pH (pHi) and cell volume. Resting pHi was 7.17 ± 0.01 in physiological medium (5% CO2–25 mM HCO3−). During carbachol (CCh) stimulation, pHi fell transiently by 0.08 ± 0.01 U concomitantly with a fall in Cl− content revealed by cell shrinkage, reflecting Cl− secretion. A subsequent alkalinization elevated pHi to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO2–HCO3−-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO3− efflux by ion substitution or exposure to the Cl− channel inhibitor niflumic acid (100 μM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na+/H+ exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1–4 and 6–9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pHi recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO3− during Ca2+-evoked fluid

A fluid secretion pathway unmasked by acinar-specific Tmem16A gene ablation in the adult mouse salivary gland

PubMed Central

Catalán, Marcelo A.; Kondo, Yusuke; Peña-Munzenmayer, Gaspar; Jaramillo, Yasna; Liu, Frances; Choi, Sooji; Crandall, Edward; Borok, Zea; Flodby, Per; Shull, Gary E.; Melvin, James E.

2015-01-01

Activation of an apical Ca2+-activated Cl− channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl− current and Cl− efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl− channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A−/−). Ca2+-dependent salivation was abolished in Tmem16A−/− mice, demonstrating that Tmem16A is obligatory for Ca2+-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A−/− mice in response to the β-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr∆F508/∆F508) or ClC-2 (Clcn2−/−) Cl− channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl− channel. Indeed, Cl− channel blockers abolished fluid secretion, indicating that Cl− channel activity is critical for IPR-stimulated secretion. These data suggest that β-adrenergic–induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A−/− mice identify Tmem16A as the Cl− channel essential for muscarinic, Ca2+-dependent fluid secretion in adult mouse salivary glands. PMID:25646474

Altered coupling of muscarinic acetylcholine receptors in pancreatic acinar carcinoma of rat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chien, J.L.; Warren, J.R.

The structure and function of muscarinic acetylcholine receptors (mAChR) in acinar carcinoma cells have been compared to mAChR in normal pancreatic acinar cells. Similar 80 kD proteins identified by SDS-PAGE of tumor and normal mAChR affinity-labeled with the muscarinic antagonist /sup 3/H-propylbenzilyl-choline mustards, and identical binding of the antagonist N-methylscopolamine to tumor and normal cells (K/sub D/approx.4x10/sup -10/ M), indicate conservation of mAChR proteins in carcinoma cells. Carcinoma mAChR display homogeneous binding of the agonists carbamylcholine (CCh), K/sub D/approx.3x10/sup -5/ M, and oxotremorine (Oxo), K/sub D/approx.x10/sup -6/ M, whereas normal cells display heterogeneous binding, with a minor component of highmore » affinity interactions for CCh, K/sub D/approx.3x10/sup -6/ M, and Oxo, K/sub D/approx.2x/sup -17/ M, and a major component of low affinity interactions for CCh, K/sub D/approx.1x10/sup -4/ M, and Oxo, K/sub D/approx.2x10/sup -5/ M. Both carcinoma and normal cells exhibit concentration-dependent CCh-stimulated increase in cytosolic free Ca/sup 2 +/, as measured by intracellular Quin 2 fluorescence and /sup 45/Ca/sup 2 +/ efflux. However, carcinoma cells demonstrate 50% maximal stimulation of intracellular Ca/sup 2 +/ release at a CCh concentration (EC/sub 50/approx.6x10/sup -7/ M) one log below that observed for normal cells. The authors propose an altered coupling of mAChR to intracellular Ca/sup 2 +/ homeostasis in carcinoma cells, which is manifest as a single activated receptor state for agonist binding, and increased sensitivity to muscarinic receptor stimulation of Ca/sup 2 +/ release.« less

Acinar Cell Carcinoma of the Pancreas: Overview of Clinicopathologic Features and Insights into the Molecular Pathology.

PubMed

La Rosa, Stefano; Sessa, Fausto; Capella, Carlo

2015-01-01

Acinar cell carcinomas (ACCs) of the pancreas are rare pancreatic neoplasms accounting for about 1-2% of pancreatic tumors in adults and about 15% in pediatric subjects. They show different clinical symptoms at presentation, different morphological features, different outcomes, and different molecular alterations. This heterogeneous clinicopathological spectrum may give rise to difficulties in the clinical and pathological diagnosis with consequential therapeutic and prognostic implications. The molecular mechanisms involved in the onset and progression of ACCs are still not completely understood, although in recent years, several attempts have been made to clarify the molecular mechanisms involved in ACC biology. In this paper, we will review the main clinicopathological and molecular features of pancreatic ACCs of both adult and pediatric subjects to give the reader a comprehensive overview of this rare tumor type.

The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum

PubMed Central

Truchan, Hilary K.; Cockburn, Chelsea L.; Hebert, Kathryn S.; Magunda, Forgivemore; Noh, Susan M.; Carlyon, Jason A.

2016-01-01

The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly

c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

PubMed

La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

2018-05-02

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Canine mammary minute oncocytomas with neuroendocrine differentiation associated with multifocal acinar cell oncocytic metaplasia.

PubMed

Nagahara, Rei; Kimura, Masayuki; Itahashi, Megu; Sugahara, Go; Kawashima, Masashi; Murayama, Hirotada; Yoshida, Toshinori; Shibutani, Makoto

2016-11-01

Two solitary and minute tumors of 1 and 1.5 mm diameter were identified by microscopy in the left fourth mammary gland of a 13-year-old female Labrador Retriever dog, in addition to multiple mammary gland tumors. The former tumors were well circumscribed and were composed of small-to-large polyhedral neoplastic oncocytes with finely granular eosinophilic cytoplasm, and were arranged in solid nests separated by fine fibrovascular septa. Scattered lumina of variable sizes containing eosinophilic secretory material were evident. Cellular atypia was minimal, and no mitotic figures were visible. One tumor had several oncocytic cellular foci revealing cellular transition, with perivascular pseudorosettes consisting of columnar epithelial cells surrounding the fine vasculature. Scattered foci of mammary acinar cell hyperplasia showing oncocytic metaplasia were also observed. Immunohistochemically, the cytoplasm of neoplastic cells of the 2 microtumors showed diffuse immunoreactivity to anti-cytokeratin antibody AE1/AE3, and finely granular immunoreactivity for 60-kDa heat shock protein, mitochondrial membrane ATP synthase complex V beta subunit, and chromogranin A. One tumor also had oncocytic cellular foci forming perivascular pseudorosettes showing cellular membrane immunoreactivity for neural cell adhesion molecule. The tumors were negative for smooth muscle actin, neuron-specific enolase, vimentin, desmin, S100, and synaptophysin. Ultrastructural observation confirmed the abundant mitochondria in the cytoplasm of both neoplastic and hyperplastic cells, the former cells also having neuroendocrine granule-like electron-dense bodies. From these results, our case was diagnosed with mammary oncocytomas accompanied by neuroendocrine differentiation. Scattered foci of mammary oncocytosis might be related to the multicentric occurrence of these oncocytomas. © 2016 The Author(s).

Fullerenol Cytotoxicity in Kidney Cells is Associated with Cytoskeleton Disruption, Autophagic Vacuole Accumulation, and Mitochondrial Dysfunction

PubMed Central

Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

2010-01-01

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C60OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials. PMID:20713077

Water permeability of acinar cell membranes in the isolated perfused rabbit mandibular salivary gland.

PubMed Central

Steward, M C; Seo, Y; Rawlings, J M; Case, R M

1990-01-01

1. The diffusive water permeability of epithelial cell membranes in the perfused rabbit mandibular salivary gland was measured at 37 degrees C by a 1H nuclear magnetic resonance relaxation method using an extracellular relaxation reagent, gadolinium diethylenetriaminepentaacetic acid (Gd(DTPA)). 2. In glands perfused with a HEPES-buffered solution containing 10 mmol l-1 Gd(DTPA), the spin-lattice (T1) relaxation of the water protons showed two exponential components. The water compartment responsible for the slower component corresponded in magnitude to 71 +/- 5% of the wet weight of the gland, and was attributed to the exchangeable intracellular water of the acinar cells. 3. The rate constant for water efflux from the cells was estimated to be 4.1 +/- 0.1 s-1 which would be consistent with a diffusive membrane permeability (Pd) of approximately 3 x 10(-3) cm s-1. Stimulation with acetylcholine (10(-6) mol l-1) did not cause any detectable change in membrane water permeability. 4. Since the basolateral membrane probably provides the main pathway for water efflux, the osmotic water permeability of this barrier (expressed per gland) was estimated to be less than 6.2 cm3 s-1. This would be insufficient to account for the generation of a near-isosmotic fluid at the flow rates observed during secretion, and suggests that a substantial fraction of the flow of water occurs via a paracellular route. PMID:1966053

Expression of alveolar type II cell markers in acinar adenocarcinomas and adenoid cystic carcinomas arising from segmental bronchi. A study in a heterotopic bronchogenic carcinoma model in dogs.

PubMed Central

TenHave-Opbroek, A. A.; Hammond, W. G.; Benfield, J. R.; Teplitz, R. L.; Dijkman, J. H.

1993-01-01

The type II alveolar epithelial cell is one of two pluripotential stem cell phenotypes in normal mammalian lung morphogenesis; cells manifesting this phenotype have been found to constitute bronchioloalveolar regions of canine adenocarcinomas. We now studied type II cell expression in canine acinar adenocarcinomas and adenoid cystic (bronchial gland) carcinomas, using the same bronchogenic carcinoma model (subcutaneous bronchial autografts treated with 3-methylcholanthrene). Distinctive features of type II cells are the approximately cuboid cell shape, large and roundish nucleus, immunofluorescent staining of the cytoplasm for the surfactant protein SP-A, and presence of multilamellar bodies or their precursory forms. Cells with these type II cell characteristics were found in the basal epithelial layer of all tumor lesions and in upper layers as far as the lumen, singly or in clusters; they were also found in early invasive carcinomatous lesions but not in bronchial glands or bronchial epithelium before carcinogen exposure. Immunoblots of tumor homogenates showed reactive proteins within size classes of SP-A (28 to 36 kd) or its dimeric form (56 to 72 kd). These findings and those previously reported are consistent with the concept that chemical carcinogenesis in the adult bronchial epithelium may lead to type II cell carcinomas of varying glandular (acinar, adenoidcystic or bronchioloalveolar) growth patterns. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 Figure 19 Figure 20 Figure 21 Figure 22 PMID:8386445

The Toxoplasma Parasitophorous Vacuole: An Evolving Host-Parasite Frontier.

PubMed

Clough, Barbara; Frickel, Eva-Maria

2017-06-01

The parasitophorous vacuole is a unique replicative niche for apicomplexan parasites, including Toxoplasma gondii. Derived from host plasma membrane, the vacuole is rendered nonfusogenic with the host endolysosomal system. Toxoplasma secretes numerous proteins to modify the forming vacuole, enable nutrient uptake, and set up mechanisms of host subversion. Here we describe the pathways of host-parasite interaction at the parasitophorous vacuole employed by Toxoplasma and host, leading to the intricate balance of host defence versus parasite survival. Copyright © 2017 Elsevier Ltd. All rights reserved.

An electron microscope study of the contractile vacuole in Tokophrya infusionum.

PubMed

RUDZINSKA, M A

1958-03-25

Contractile vacuoles are organelles that collect fluid from the cytoplasm and expel it to the outside. After each discharge (systole), they appear again and expand (diastole). They are widely distributed among Protozoa, and have been found also in some fresh water algae, sponges, and recently in some blood cells of the frog, guinea pig, and man. In spite of the extensive work on the contractile vacuole, very little is known concerning its mode of operation. An electron microscope study of a suctorian Tokophrya infusionum provided an opportunity to study thin sections of contractile vacuoles, and in these some structures were found which could be part of a mechanism for the systolic and diastolic motions the organelle displays. In Tokophrya, as in Suctoria and Ciliata in general, the contractile vacuole has a permanent canal connecting it with the outside. The canal appears to have a very elaborate structure and is composed of three parts: (1) a pore; (2) a channel; and (3) a narrow tubule located in a papilla protruding into the cavity of the contractile vacuole. Whereas the pore and channel have fixed dimensions and are permanently widely open, the tubule has a changeable diameter. At diastole it is so narrow (about 25 to 30 mmicro in diameter) that it could be regarded as closed, while at systole it is widely open. It is assumed that the change in diameter is due to the contraction of numerous fine fibrils (about 180 A thick) which are radially disposed around the canal in form of a truncated cone, with its tip at the channel, and its base at the vacuolar membrane. It seems most probable that the broadening of the tubule results in discharge of the content of the contractile vacuole. In the vicinity of the very thin limiting vacuolar membrane, small vesicles and canaliculi of the endoplasmic reticulum, very small dense particles, and mitochondria may be found. In addition, rows of closely packed vesicles are present in this region, and in other parts of the

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism.

PubMed

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R; Raikhel, Natasha V

2015-01-06

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

Esculetin and esculin (esculetin 6-O-glucoside) occur as inclusions and are differentially distributed in the vacuole of palisade cells in Fraxinus ornus leaves: a fluorescence microscopy analysis.

PubMed

Tattini, Massimiliano; Di Ferdinando, Martina; Brunetti, Cecilia; Goti, Andrea; Pollastri, Susanna; Bellasio, Chandra; Giordano, Cristiana; Fini, Alessio; Agati, Giovanni

2014-11-01

The location of individual coumarins in leaves of Fraxinus ornus acclimated at full solar irradiance was estimated using their specific UV- and fluorescence spectral features. Using a combination of UV-induced fluorescence and blue light-induced fluorescence of tissues stained with diphenylborinic acid 2-amino-ethylester, in wide field or confocal laser scanning microscopy, we were able to visualize the distribution of esculetin and esculetin 6-O-glucoside (esculin) in palisade cells. Coumarins are not uniformly distributed in the cell vacuole, but accumulate mostly in the adaxial portion of palisade cells. Our study indeed shows, for the first time, that coumarins in palisade cells accumulate as vacuolar inclusions, as previously reported in the pertinent literature only for anthocyanins. Furthermore, esculetin and esculin have a different vacuolar distribution: esculetin largely predominates in the first 15 μm from the adaxial epidermis. This leads to hypothesize for esculetin and esculin different transport mechanisms from the endoplasmic reticulum to the vacuole as well as potentially different roles in photoprotection. Our study open to new experiments aimed at exploring the mechanisms that deliver coumarins to the vacuole using different fluorescence signatures of coumarin aglycones and coumarin glycosides. Copyright © 2014 Elsevier B.V. All rights reserved.

Rimmed vacuoles in Becker muscular dystrophy have similar features with inclusion myopathies.

PubMed

Momma, Kazunari; Noguchi, Satoru; Malicdan, May Christine V; Hayashi, Yukiko K; Minami, Narihiro; Kamakura, Keiko; Nonaka, Ikuya; Nishino, Ichizo

2012-01-01

Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

LMW-E/CDK2 Deregulates Acinar Morphogenesis, Induces Tumorigenesis, and Associates with the Activated b-Raf-ERK1/2-mTOR Pathway in Breast Cancer Patients

PubMed Central

Duong, MyLinh T.; Akli, Said; Wei, Caimiao; Wingate, Hannah F.; Liu, Wenbin; Lu, Yiling; Yi, Min; Mills, Gordon B.; Hunt, Kelly K.; Keyomarsi, Khandan

2012-01-01

Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW

Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

PubMed

Ding, Li; Liou, Geou-Yarh; Schmitt, Daniel M; Storz, Peter; Zhang, Jin-San; Billadeau, Daniel D

2017-09-01

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

PubMed Central

Shapiro, John P; Komar, Hannah M; Hancioglu, Baris; Yu, Lianbo; Jin, Ming; Ogata, Yuko; Hart, Phil A; Cruz-Monserrate, Zobeida; Lesinski, Gregory B; Conwell, Darwin L

2017-01-01

Objectives: Chronic pancreatitis (CP) is characterized by inflammation and fibrosis of the pancreas, leading to pain, parenchymal damage, and loss of exocrine and endocrine function. There are currently no curative therapies; diagnosis remains difficult and aspects of pathogenesis remain unclear. Thus, there is a need to identify novel biomarkers to improve diagnosis and understand pathophysiology. We hypothesize that pancreatic acinar regions contain proteomic signatures relevant to disease processes, including secreted proteins that could be detected in biofluids. Methods: Acini from pancreata of mice injected with or without caerulein were collected using laser capture microdissection followed by mass spectrometry analysis. This protocol enabled high-throughput analysis that captured altered protein expression throughout the stages of CP. Results: Over 2,900 proteins were identified, whereas 331 were significantly changed ≥2-fold by mass spectrometry spectral count analysis. Consistent with pathogenesis, we observed increases in proteins related to fibrosis (e.g., collagen, P<0.001), several proteases (e.g., trypsin 1, P<0.001), and altered expression of proteins associated with diminished pancreas function (e.g., lipase, amylase, P<0.05). In comparison with proteomic data from a public data set of CP patients, a significant correlation was observed between proteomic changes in tissue from both the caerulein model and CP patients (r=0.725, P<0.001). CONCLUSIONS: This study illustrates the ability to characterize proteome changes of acinar cells isolated from pancreata of caerulein-treated mice and demonstrates a relationship between signatures from murine and human CP. PMID:28406494

A bicarbonate- and weak acid-permeable chloride conductance controlled by cytosolic Ca2+ and ATP in rat submandibular acinar cells.

PubMed

Ishikawa, T

1996-09-01

A Ca(2+)-activated Cl- conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mM ATP and 1 microM free Ca2+ and bathed in N-methyl-D-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nM or less than 1 nM free Ca2+ strongly reduced the Cl- currents, indicating the currents were Ca(2+)-dependent. Relaxation analysis of the "on" currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl- channels was: NO3- (2.00) > I- (1.85) > or = Br- (1.69) > Cl- (1.00) > bicarbonate (0.77) > or = acetate (0.70) > propionate (0.41) > > glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mM, the Ca(2+)-dependency of the Cl- current amplitude shifted to lower free Ca2+ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-gamma S (2 mM) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mM). The addition of the calmodulin inhibitors trifluoperazine (100 microM) or calmidazolium (25 microM) to the bath solution and the inclusion of KN-62 (1 microM), a specific inhibitor of calmodulin kinase, or staurosporin (10 nM), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca(2+)-activated Cl- currents. This suggests that Ca2+/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca(2+)-activated Cl- currents. The outward Cl- currents at +69 mV were inhibited by NPPB (100 microM), IAA-94 (100 microM), DIDS (0.03-1 mM), 9-AC (300 microM and 1 mM) and DPC (1 mM), whereas the inward currents at -101 mV were not. These results demonstrate the presence of a

Heterologous desensitization of muscarinic receptors by P2Z purinoceptors in rat parotid acinar cells.

PubMed

Fukushi, Y

1999-01-01

We studied the heterologous desensitization of muscarinic receptors by ATP in fura-2-loaded rat parotid acinar cells. Exposure to ATP or 3'-o-(4-benzoyl) benzoyl-ATP shortened the duration and decreased the magnitude of acetylcholine-induced Ca2+ release from intracellular Ca2+ stores in a dose-dependent manner. The shortening was observed only in an early stage of desensitization (within 20 s), whereas the decrease in the magnitude of the response was dependent upon the time the cells were exposed to the nucleotides. Atropine induced a profound shortening during the progressive decrease in the magnitude of acetylcholine-induced Ca2+ release. 3'-o-(4-Benzoyl) benzoyl-ATP did not induce an increase in the cytosolic Ca2+ concentration when the cells were incubated in the Ca2+- and Na+-free medium, but it did induce a strong desensitization of muscarinic receptors. The specific protein kinase C inhibitor bisindoylmaleimide resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. Phorbol 12-myristate 13-acetate potentiated the desensitization of muscarinic receptors. Ceramides that prevent the activation of phospholipase D resensitized the 3'-o-(4-benzoyl) benzoyl-ATP-treated muscarinic receptors. These results suggest that ATP, acting through P2Z purinoceptor-mediated phospholipase D, may produce a Ca2+-independent protein kinase C. Heterologous desensitization of muscarinic receptors by protein kinase C may shorten the duration and decrease the magnitude of acetylcholine-induced Ca2+ release.

Protease activation during in vivo pancreatitis is dependent on calcineurin activation.

PubMed

Shah, Ahsan U; Sarwar, Amna; Orabi, Abrahim I; Gautam, Samir; Grant, Wayne M; Park, Alexander J; Shah, Adnan U; Liu, Jun; Mistry, Pramod K; Jain, Dhanpat; Husain, Sohail Z

2009-11-01

The premature activation of digestive proenzymes, specifically proteases, within the pancreatic acinar cell is an early and critical event during acute pancreatitis. Our previous studies demonstrate that this activation requires a distinct pathological rise in cytosolic Ca(2+). Furthermore, we have shown that a target of aberrant Ca(2+) in acinar cells is the Ca(2+)/calmodulin-dependent phosphatase calcineurin (PP2B). In this study, we hypothesized that PP2B mediates in vivo protease activation and pancreatitis severity. To test this, pancreatitis was induced in mice over 8 h by administering hourly intraperitoneal injections of the cholecystokinin analog caerulein (50 microg/kg). Treatment with the PP2B inhibitor FK506 at 1 and 8 h after pancreatitis induction reduced trypsin activities by greater than 50% (P < 0.005). Serum amylase and IL-6 was reduced by 86 and 84% relative to baseline (P < 0.0005) at 8 h, respectively. Histological severity of pancreatitis, graded on the basis of pancreatic edema, acinar cell vacuolization, inflammation, and apoptosis, was reduced early in the course of pancreatitis. Myeloperoxidase activity from both pancreas and lung was reduced by 93 and 83% relative to baseline, respectively (P < 0.05). These data suggest that PP2B is an important target of the aberrant acinar cell Ca(2+) rise associated with pathological protease activation and pancreatitis.

Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation

PubMed Central

Okuda, Kendi; Tong, Mei; Dempsey, Brian; Moore, Kathryn J.; Gazzinelli, Ricardo T.; Silverman, Neal

2016-01-01

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection. PMID:27280707

Delivery of Prolamins to the Protein Storage Vacuole in Maize Aleurone Cells[W

PubMed Central

Reyes, Francisca C.; Chung, Taijoon; Holding, David; Jung, Rudolf; Vierstra, Richard; Otegui, Marisa S.

2011-01-01

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals. PMID:21343414

Particle dynamics and deposition in true-scale pulmonary acinar models.

PubMed

Fishler, Rami; Hofemeier, Philipp; Etzion, Yael; Dubowski, Yael; Sznitman, Josué

2015-09-11

Particle transport phenomena in the deep alveolated airways of the lungs (i.e. pulmonary acinus) govern deposition outcomes following inhalation of hazardous or pharmaceutical aerosols. Yet, there is still a dearth of experimental tools for resolving acinar particle dynamics and validating numerical simulations. Here, we present a true-scale experimental model of acinar structures consisting of bifurcating alveolated ducts that capture breathing-like wall motion and ensuing respiratory acinar flows. We study experimentally captured trajectories of inhaled polydispersed smoke particles (0.2 to 1 μm in diameter), demonstrating how intrinsic particle motion, i.e. gravity and diffusion, is crucial in determining dispersion and deposition of aerosols through a streamline crossing mechanism, a phenomenon paramount during flow reversal and locally within alveolar cavities. A simple conceptual framework is constructed for predicting the fate of inhaled particles near an alveolus by identifying capture and escape zones and considering how streamline crossing may shift particles between them. In addition, we examine the effect of particle size on detailed deposition patterns of monodispersed microspheres between 0.1-2 μm. Our experiments underline local modifications in the deposition patterns due to gravity for particles ≥0.5 μm compared to smaller particles, and show good agreement with corresponding numerical simulations.

Particle dynamics and deposition in true-scale pulmonary acinar models

PubMed Central

Fishler, Rami; Hofemeier, Philipp; Etzion, Yael; Dubowski, Yael; Sznitman, Josué

2015-01-01

Particle transport phenomena in the deep alveolated airways of the lungs (i.e. pulmonary acinus) govern deposition outcomes following inhalation of hazardous or pharmaceutical aerosols. Yet, there is still a dearth of experimental tools for resolving acinar particle dynamics and validating numerical simulations. Here, we present a true-scale experimental model of acinar structures consisting of bifurcating alveolated ducts that capture breathing-like wall motion and ensuing respiratory acinar flows. We study experimentally captured trajectories of inhaled polydispersed smoke particles (0.2 to 1 μm in diameter), demonstrating how intrinsic particle motion, i.e. gravity and diffusion, is crucial in determining dispersion and deposition of aerosols through a streamline crossing mechanism, a phenomenon paramount during flow reversal and locally within alveolar cavities. A simple conceptual framework is constructed for predicting the fate of inhaled particles near an alveolus by identifying capture and escape zones and considering how streamline crossing may shift particles between them. In addition, we examine the effect of particle size on detailed deposition patterns of monodispersed microspheres between 0.1–2 μm. Our experiments underline local modifications in the deposition patterns due to gravity for particles ≥0.5 μm compared to smaller particles, and show good agreement with corresponding numerical simulations. PMID:26358580

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

DOE PAGES

Li, Ruixi; Sun, Ruobai; Hicks, Glenn R.; ...

2014-12-22

The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red stainingmore » suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.« less

Effects of Erdosteine on Experimental Acute Pancreatitis Model.

PubMed

Karapolat, Banu; Karapolat, Sami; Gurleyik, Emin; Yasar, Mehmet

2017-10-01

To create acute pancreatitis condition experimentally in rats using cerulein, and to reveal histopathological effects in pancreatic tissue with erdosteine. An experimental study. Department of General Surgery, Duzce University, Turkey, from June to October 2014. Thirty male Wistar albino rats were divided into three groups. No procedures were applied to Group 1. The rats in Group 2 and Group 3 were injected cerulein, to establish an experimental pancreatitis model and the blood amylase and lipase values were examined. The rats in Group 3 were given 10 mg/kg erdosteine. This treatment was continued for another 2 days and the rats were sacrificed. The pancreatic tissues were examined histopathologically for edema, inflammation, acinar necrosis, fat necrosis, and vacuolization. The lipase and amylase values and the histopathological examination of pancreatic tissues evidenced that the experimental acute pancreatitis model was established and edema, inflammation, acinar necrosis, fat necrosis, and vacuolization were observed in the pancreatic tissues. The statistical results suggest that erdosteine can decrease the edema, inflammation, acinar necrosis, fat necrosis and vacuolization scores in the tissues. The severity of acute pancreatitis, induced by cerulein in rats, is reduced with the use of erdosteine.

STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells

PubMed Central

Guo, Liang; Lichten, Louis A.; Ryu, Moon-Suhn; Liuzzi, Juan P.; Wang, Fudi; Cousins, Robert J.

2010-01-01

The exocrine pancreas plays an important role in endogenous zinc loss by regulating excretion into the intestinal tract and hence influences the dietary zinc requirement. The present experiments show that the zinc transporter ZnT2 (Slc30a2) is localized to the zymogen granules and that dietary zinc restriction in mice decreased the zinc concentration of zymogen granules and ZnT2 expression. Excess zinc given orally increased ZnT2 expression and was associated with increased pancreatic zinc accumulation. Rat AR42J acinar cells when induced into a secretory phenotype, using the glucocorticoid analog dexamethasone (DEX), exhibited increased ZnT2 expression and labile zinc as measured with a fluorophore. DEX administrated to mice also induced ZnT2 expression that accompanied a reduction of the pancreatic zinc content. ZnT2 promoter analyses identified elements required for responsiveness to zinc and DEX. Zinc regulation was traced to a MRE located downstream from the ZnT2 transcription start site. Responsiveness to DEX is produced by two upstream STAT5 binding sites that require the glucocorticoid receptor for activation. ZnT2 knockdown in the AR42J cells using siRNA resulted in increased cytoplasmic zinc and decreased zymogen granule zinc that further demonstrated that ZnT2 may mediate the sequestration of zinc into zymogen granules. We conclude, based upon experiments with intact mice and pancreatic acinar cells in culture, that ZnT2 participates in zinc transport into pancreatic zymogen granules through a glucocorticoid pathway requiring glucocorticoid receptor and STAT5, and zinc-regulated signaling pathways requiring MTF-1. The ZnT2 transporter appears to function in a physiologically responsive manner involving entero-pancreatic zinc trafficking. PMID:20133611

Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles

PubMed Central

Cna'ani, Alon; Shavit, Reut; Ravid, Jasmin; Aravena-Calvo, Javiera; Skaliter, Oded; Masci, Tania; Vainstein, Alexander

2017-01-01

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography–mass spectrometry (GC–MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger β-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC–MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites. PMID:29163617

Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

PubMed Central

Irani, Niloufer G; Grotewold, Erich

2005-01-01

Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS) cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI)-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin. PMID:15907203

Purification and proteomics of pathogen-modified vacuoles and membranes

PubMed Central

Herweg, Jo-Ana; Hansmeier, Nicole; Otto, Andreas; Geffken, Anna C.; Subbarayal, Prema; Prusty, Bhupesh K.; Becher, Dörte; Hensel, Michael; Schaible, Ulrich E.; Rudel, Thomas; Hilbi, Hubert

2015-01-01

Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e., the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania, and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation. PMID:26082896

Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles

PubMed Central

Nolan, Sabrina J.; Luechtefeld, Thomas

2015-01-01

Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths. PMID:25750213

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase in nuclei and rimmed vacuoles of muscle fibers in DMRV (distal myopathy with rimmed vacuoles).

PubMed

Ishihara, Shoichiro; Tomimitsu, Hiroyuki; Fujigasaki, Hiroto; Saito, Fumiaki; Mizusawa, Hidehiro

2008-03-01

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is a key molecule in the pathogenesis of distal myopathy with rimmed vacuoles (DMRV) and hereditary inclusion body myopathy (HIBM) and almost all such patients have some mutations in GNE. However, subcellular localization of GNE and the mechanism of muscular damage have not been clarified. A rabbit polyclonal antibody for GNE was prepared. Immunohistochemistry was performed using anti-GNE and anti-nuclear protein antibodies. Western blotting with subcellular fractionated proteins was performed to determine subcellular localization of GNE. The sizes of myonuclei were quantified in muscle biopsies from patients with DMRV and amyotrophic lateral sclerosis (ALS). In DMRV muscles, immunohistochemistry identified GNE in sarcoplasm and specifically in myonuclei and rimmed vacuoles (RV). Nuclear proteins were also found in RVs. Immunohistochemistry showed colocalization of GNE and emerin in C2C12 cells. Western blotting revealed the presence of GNE in nuclear fractions of human embryonic kidney (HEK) 293T cells. The mean size of myonuclei of DMRV was significantly larger than that of ALS. GNE is present in myonuclei near nuclear membrane. Our results suggest that myonuclei are involved in RV formation in DMRV, and that mutant GNE in myonuclei seems to play some role in this process.

Cholecystokinin-8-induced hypoplasia of the rat pancreas: influence of nitric oxide on cell proliferation and programmed cell death.

PubMed

Trulsson, Lena M; Gasslander, Thomas; Svanvik, Joar

2004-10-01

The background of cholecystokinin-8 (CCK-8)-induced hypoplasia in the pancreas is not known. In order to increase our understanding we studied the roles of nitric oxide and NF-kappaB in rats. CCK-8 was injected for 4 days, in a mode known to cause hypoplasia, and the nitric oxide formation was either decreased by means of N(omega)-nitro-L-arginine (L-NNA) or increased by S-nitroso-N-acetylpencillamine (SNAP). The activation of NF-kappaB was quantified by ELISA detection, apoptosis with caspase-3 and histone-associated DNA-fragmentation and mitotic activity in the acinar, centroacinar and ductal cells were visualized by the incorporation of [(3)H]-thymidine. Pancreatic histology and weight as well as protein- and DNA contents were also studied. Intermittent CCK injections reduced pancreatic weight, protein and DNA contents and increased apoptosis, acinar cell proliferation and nuclear factor kappaB (NF-kappaB) activation. It also caused vacuolisation of acinar cells. The inhibition of endogenous nitric oxide formation by L-NNA further increased apoptosis and NF-kappaB activation but blocked the increased proliferation and vacuolisation of acinar cells. The DNA content was not further reduced. SNAP given together with CCK-8 increased apoptosis and other pathways of cell death, raised proliferation of acinar cells and strongly reduced the DNA content in the pancreas. Histological examination showed no inflammation in any group. We conclude that during CCK-8-induced pancreatic hypoplasia, endogenously formed nitric oxide suppresses apoptosis but increases cell death along non-apoptotic pathways and stimulates regeneration of acinar cells. Exogenous nitric oxide enhances the acinar cell turnover by increasing both apoptotic and non-apoptotic cell death and cell renewal. In this situation NF-kappaB activation seems not to inhibit apoptosis nor promote cell proliferation.

The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

PubMed Central

Geiser, Jim; De Lisle, Robert C.; Andrews, Glen K.

2013-01-01

Background ZIP5 localizes to the baso-lateral membranes of intestinal enterocytes and pancreatic acinar cells and is internalized and degraded coordinately in these cell-types during periods of dietary zinc deficiency. These cell-types are thought to control zinc excretion from the body. The baso-lateral localization and zinc-regulation of ZIP5 in these cells are unique among the 14 members of the Slc39a family and suggest that ZIP5 plays a role in zinc excretion. Methods/Principal Findings We created mice with floxed Zip5 genes and deleted this gene in the entire mouse or specifically in enterocytes or acinar cells and then examined the effects on zinc homeostasis. We found that ZIP5 is not essential for growth and viability but total knockout of ZIP5 led to increased zinc in the liver in mice fed a zinc-adequate (ZnA) diet but impaired accumulation of pancreatic zinc in mice fed a zinc-excess (ZnE) diet. Loss-of-function of enterocyte ZIP5, in contrast, led to increased pancreatic zinc in mice fed a ZnA diet and increased abundance of intestinal Zip4 mRNA. Finally, loss-of-function of acinar cell ZIP5 modestly reduced pancreatic zinc in mice fed a ZnA diet but did not impair zinc uptake as measured by the rapid accumulation of 67zinc. Retention of pancreatic 67zinc was impaired in these mice but the absence of pancreatic ZIP5 sensitized them to zinc-induced pancreatitis and exacerbated the formation of large cytoplasmic vacuoles containing secretory protein in acinar cells. Conclusions These studies demonstrate that ZIP5 participates in the control of zinc excretion in mice. Specifically, they reveal a paramount function of intestinal ZIP5 in zinc excretion but suggest a role for pancreatic ZIP5 in zinc accumulation/retention in acinar cells. ZIP5 functions in acinar cells to protect against zinc-induced acute pancreatitis and attenuate the process of zymophagy. This suggests that it may play a role in autophagy. PMID:24303081

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

PubMed

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

PubMed Central

Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. PMID:25713145

Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

PubMed

Bruno, P; Calastretti, A; Priulla, M; Asnaghi, L; Scarlatti, F; Nicolin, A; Canti, G

2007-10-01

Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

PubMed

Vinayek, R; Patto, R J; Menozzi, D; Gregory, J; Mrozinski, J E; Jensen, R T; Gardner, J D

1993-03-10

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

A Rab-centric perspective of bacterial pathogen-occupied vacuoles.

PubMed

Sherwood, Racquel Kim; Roy, Craig R

2013-09-11

The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles. Copyright © 2013 Elsevier Inc. All rights reserved.

pH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

PubMed

Funamoto, Rintaro; Saito, Katsuharu; Oyaizu, Hiroshi; Aono, Toshihiro; Saito, Masanori

2015-01-01

Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

High-Spin Ferric Ions in Saccharomyces cerevisiae Vacuoles Are Reduced to the Ferrous State during Adenine-Precursor Detoxification

PubMed Central

2015-01-01

The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) FeIII complexes with polyphosphate-related ligands. Some FeIII oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS FeIII. Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM FeIII citrate) accumulated two major NHHS FeII species as the vacuolar NHHS FeIII species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS FeII complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS FeIII with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS FeIII species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated. PMID:24919141

Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells.

PubMed

Kubisch, Constanze H; Logsdon, Craig D

2007-06-01

Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV-180 (10 nM-10 microM) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas.

New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists.

PubMed

Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

2013-01-01

While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. © 2013, Elsevier Inc. All Rights Reserved.

Three dimensional cultures: a tool to study normal acinar architecture vs. malignant transformation of breast cells.

PubMed

Pal, Anupama; Kleer, Celina G

2014-04-25

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior(1). However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex vivo system(2,3). Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D(4). 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved(3). One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D(6,7). Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during

Acinar cell carcinoma of the pancreas presenting as diffuse pancreatic enlargement: Two case reports and literature review.

PubMed

Luo, Yaping; Hu, Guilan; Ma, Yanru; Guo, Ning; Li, Fang

2017-09-01

Pancreatic acinar cell carcinoma (ACC) is a rare malignant tumor of exocrine pancreas. It is typically a well-marginated large solid mass arising in a certain aspect of the pancreas. Diffuse involvement of ACC in the pancreas is very rare, and may simulate pancreatitis in radiological findings. We report 2 cases of ACC presenting as diffuse enlargement of the pancreas due to tumor involvement without formation of a distinct mass. The patients consisted of a 41-year-old man with weight loss and a 77-year-old man who was asymptomatic. Computed tomography (CT) and 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET)/CT showed diffuse enlargement of the pancreas forming a sausage-like shape with homogenously increased FDG activity. Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) biopsy of the pancreatic lesion was performed. Histopathology results from the pancreas confirmed the diagnosis of pancreatic ACC. Because diffuse enlargement of the pancreas is a common imaging feature of pancreatitis, recognition of this rare morphologic pattern of ACC is important for radiological diagnosis of this tumor.

Action of cholecystokinin and cholinergic agents on calcium transport in isolated pancreatic acinar cells.

PubMed Central

Gardner, J D; Conlon, T P; Kleveman, H L; Adams, T D; Ondetti, M A

1975-01-01

COOH-terminal octapeptide of cholecystokinin (CCK-octapeptide) and the cholinergic agent carbamylcholine each produced a fourfold stimulation of calcium outflux in guinea pig isolated pancreatic acinar cells. Neither agent altered calcium influx. Stimulation of calcium outflux was rapid and specific, was abolished by reducing the incubation temperature to 4 degrees C, and was a saturable function of the secretagogue concentration. The concentrations of CCK-octapeptide and carbamylcholine that produced half-maximal stimulation of calcium outflux were 3.1 x 10(-10) M and 4.9 x 10(-5) M, respectively. The cholinergic antagonist antropine competitively inhibited carbamylcholine stimulation of calcium outflux but did not alter stimulation produced by CCK-octapeptide. Stimulation of calcium outflux by maximal concentrations of carbamycholine plus CCK-octapeptide was the same as that produced by a maximal concentration of either agent alone.Calcium outflux became refractory to stimulation by secretagogues, and incubation with either CCK-ostapeptide or carbamylcholine produced a refractoriness to both agents. The relative potencies with CCK and its related fragments stimulated calcium outflux were CCK-octapeptide greater than heptapeptide greater than CCK greater than hexapeptide = gastrin. Secretin, glucagon, and vasoactive intestinal peptide, at concentrations as high as 10(-5) M, failed to alter calcium outflux and did not affect stimulation by CCK-octapeptide or by carbamycholine. Images PMID:1150877

Steady streaming: A key mixing mechanism in low-Reynolds-number acinar flows

PubMed Central

Kumar, Haribalan; Tawhai, Merryn H.; Hoffman, Eric A.; Lin, Ching-Long

2011-01-01

Study of mixing is important in understanding transport of submicron sized particles in the acinar region of the lung. In this article, we investigate transport in view of advective mixing utilizing Lagrangian particle tracking techniques: tracer advection, stretch rate and dispersion analysis. The phenomenon of steady streaming in an oscillatory flow is found to hold the key to the origin of kinematic mixing in the alveolus, the alveolar mouth and the alveolated duct. This mechanism provides the common route to folding of material lines and surfaces in any region of the acinar flow, and has no bearing on whether the geometry is expanding or if flow separates within the cavity or not. All analyses consistently indicate a significant decrease in mixing with decreasing Reynolds number (Re). For a given Re, dispersion is found to increase with degree of alveolation, indicating that geometry effects are important. These effects of Re and geometry can also be explained by the streaming mechanism. Based on flow conditions and resultant convective mixing measures, we conclude that significant convective mixing in the duct and within an alveolus could originate only in the first few generations of the acinar tree as a result of nonzero inertia, flow asymmetry, and large Keulegan–Carpenter (KC) number. PMID:21580803

Cellular vacuolation and mitochondrial-associated factors induced by Clostridium perfringens epsilon toxin detected using acoustic flow cytometry.

PubMed

Ferrarezi, Marina C; Curci, Vera C L M; Cardoso, Tereza C

2013-12-01

Epsilon toxin (ETX) produced by Clostridium perfringens types B and D is a potent toxin that is responsible for fatal enterotoxaemia. In vitro, ETX, which is considered as a pore-forming toxin, forms a heptamer in Madin-Darby canine kidney (MDCK) cell membranes, which is considered to be a pre-pore stage. After binding of the ETX, vacuoles inside cell cytoplasm are produced. ETX causes decreased levels of essential coenzymes required for host cell energy. Here, we optimized and applied acoustic flow cytometry analysis in order to gain further insight into ETX-pathogenesis. Using acoustic flow cytometer analysis, which considered highly sensitive, ETX-exposed MDCK cells revealed mitochondrial membrane decreases followed by 25.48% and 45.45% of the exposed cells expressing the Bax and BCL-2 proteins at a pre-pore stage, respectively. These results together with high cytotoxicity and visualization of cell vacuoles, demonstrates that acoustic flow cytometry analysis potentially represents an effective tool to study ETX pathogenesis. Copyright © 2013. Published by Elsevier Ltd.

β-Cell regeneration through the transdifferentiation of pancreatic cells: Pancreatic progenitor cells in the pancreas.

PubMed

Kim, Hyo-Sup; Lee, Moon-Kyu

2016-05-01

Pancreatic progenitor cell research has been in the spotlight, as these cells have the potential to replace pancreatic β-cells for the treatment of type 1 and 2 diabetic patients with the absence or reduction of pancreatic β-cells. During the past few decades, the successful treatment of diabetes through transplantation of the whole pancreas or isolated islets has nearly been achieved. However, novel sources of pancreatic islets or insulin-producing cells are required to provide sufficient amounts of donor tissues. To overcome this limitation, the use of pancreatic progenitor cells is gaining more attention. In particular, pancreatic exocrine cells, such as duct epithelial cells and acinar cells, are attractive candidates for β-cell regeneration because of their differentiation potential and pancreatic lineage characteristics. It has been assumed that β-cell neogenesis from pancreatic progenitor cells could occur in pancreatic ducts in the postnatal stage. Several studies have shown that insulin-producing cells can arise in the duct tissue of the adult pancreas. Acinar cells also might have the potential to differentiate into insulin-producing cells. The present review summarizes recent progress in research on the transdifferentiation of pancreatic exocrine cells into insulin-producing cells, especially duct and acinar cells.

Amino acid sequence and the cellular location of the Na(+)-dependent D-glucose symporters (SGLT1) in the ovine enterocyte and the parotid acinar cell.

PubMed Central

Tarpey, P S; Wood, I S; Shirazi-Beechey, S P; Beechey, R B

1995-01-01

The Na(+)-dependent D-glucose symporter has been shown to be located on the basolateral domain of the plasma membrane of ovine parotid acinar cells. This is in contrast to the apical location of this transporter in the ovine enterocyte. The amino acid sequences of these two proteins have been determined. They are identical. The results indicated that the signals responsible for the differential targeting of these two proteins to the apical and the basal domains of the plasma membrane are not contained within the primary amino acid sequence. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7492327

LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole.

PubMed

Schulze-Luehrmann, Jan; Eckart, Rita A; Ölke, Martha; Saftig, Paul; Liebler-Tenorio, Elisabeth; Lührmann, Anja

2016-02-01

The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii. © 2015 John Wiley & Sons Ltd.

New Insights into the Roles of Acidocalcisomes and the Contractile Vacuole Complex in Osmoregulation in Protists

PubMed Central

Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

2013-01-01

While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. PMID:23890380

Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

PubMed Central

Colombo, María Isabel; García Véscovi, Eleonora

2011-01-01

Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell. PMID:21901159

Tumour necrosis factor α secretion induces protease activation and acinar cell necrosis in acute experimental pancreatitis in mice.

PubMed

Sendler, Matthias; Dummer, Annegret; Weiss, Frank U; Krüger, Burkhard; Wartmann, Thomas; Scharffetter-Kochanek, Karin; van Rooijen, Nico; Malla, Sudarshan Ravi; Aghdassi, Ali; Halangk, Walter; Lerch, Markus M; Mayerle, Julia

2013-03-01

Acute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory cells, whose systemic action ultimately determines the disease severity. In the present work the opposite mechanism is investigated; that is, whether and how inflammatory cells can activate intracellular proteases. Using mice either deficient for the CD18-α subunit of the membrane attack complex-1 (MAC-1) complex or tumour necrosis factor (TNF)α, as well as after depletion of leucocyte subpopulations, pancreatitis was induced by 7-hourly caerulein injections (50 μg/kg, intraperitoneally). Pancreatic acini were coincubated in vitro from wild-type and cathepsin-B-deficient animals with phorbol-12-myristate-13-acetate (PMA)-activated neutrophils and macrophages, caerulein or TNFα, and activities of trypsin, cathepsin-B and caspase-3 were measured, as well as necrosis using fluorogenic substrates. TNFα was inhibited with monospecific antibodies. Deletion of CD18 prevented transmigration of leucocytes into the pancreas during pancreatitis, greatly reduced disease severity and abolished digestive protease activation. Depletion of neutrophils and macrophages equally reduced premature trypsinogen activation and disease severity. In vitro activated neutrophils and macrophages directly induced premature protease activation and cell death in pancreatic acini and stimulation of acini with TNFα induced caspase-3 activation and necrosis via a cathepsin-B and calcium-dependent mechanism. Neutralising antibodies against TNFα and genetic deletion of TNFα prevented leucocyte-induced trypsin activity and necrosis in isolated acini. The soluble inflammatory cell mediator TNFα directly induces premature protease activation and necrosis in pancreatic acinar cells. This activation depends on calcium and cathepsin-B activity. The findings

Multinodular and vacuolating neuronal tumors in epilepsy: dysplasia or neoplasia?

PubMed Central

Liu, Joan; Bongaarts, Anika; Reinten, Roy J.; Paradiso, Beatrice; Jäger, Hans Rolf; Reeves, Cheryl; Somani, Alyma; An, Shu; Marsdon, Derek; McEvoy, Andrew; Miserocchi, Anna; Thorne, Lewis; Newman, Fay; Bucur, Sorin; Honavar, Mrinalini; Jacques, Tom; Aronica, Eleonora

2017-01-01

Abstract Multinodular and vacuolating neuronal tumor (MVNT) is a new pattern of neuronal tumour included in the recently revised WHO 2016 classification of tumors of the CNS. There are 15 reports in the literature to date. They are typically associated with late onset epilepsy and a neoplastic vs. malformative biology has been questioned. We present a series of ten cases and compare their pathological and genetic features to better characterized epilepsy‐associated malformations including focal cortical dysplasia type II (FCDII) and low‐grade epilepsy‐associated tumors (LEAT). Clinical and neuroradiology data were reviewed and a broad immunohistochemistry panel was applied to explore neuronal and glial differentiation, interneuronal populations, mTOR pathway activation and neurodegenerative changes. Next generation sequencing was performed for targeted multi‐gene analysis to identify mutations common to epilepsy lesions including FCDII and LEAT. All of the surgical cases in this series presented with seizures, and were located in the temporal lobe. There was a lack of any progressive changes on serial pre‐operative MRI and a mean age at surgery of 45 years. The vacuolated cells of the lesion expressed mature neuronal markers (neurofilament/SMI32, MAP2, synaptophysin). Prominent labelling of the lesional cells for developmentally regulated proteins (OTX1, TBR1, SOX2, MAP1b, CD34, GFAPδ) and oligodendroglial lineage markers (OLIG2, SMI94) was observed. No mutations were detected in the mTOR pathway genes, BRAF, FGFR1 or MYB. Clinical, pathological and genetic data could indicate that MVNT aligns more with a malformative lesion than a true neoplasm with origin from a progenitor neuro‐glial cell type showing aberrant maturation. PMID:28833756

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

PubMed

Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

2017-06-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

'Candidatus Liberibacter asiaticus' Accumulates inside Endoplasmic Reticulum Associated Vacuoles in the Gut Cells of Diaphorina citri.

PubMed

Ghanim, Murad; Achor, Diann; Ghosh, Saptarshi; Kontsedalov, Svetlana; Lebedev, Galina; Levy, Amit

2017-12-05

Citrus greening disease known also as Huanglongbing (HLB) caused by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' (CLas) has resulted in tremendous losses and the death of millions of trees worldwide. CLas is transmitted by the Asian citrus psyllid Diaphorina citri. The closely-related bacteria 'Candidatus Liberibacter solanacearum' (CLso), associated with vegetative disorders in carrots, is transmitted by the carrot psyllid Bactericera trigonica. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is limited. It was recently reported that CLas induced changes in the nuclear architecture, and activated programmed cell death, in D. citri midgut cells. Here, we used electron and fluorescent microscopy and show that CLas induces the formation of endoplasmic reticulum (ER)-associated bodies. The bacterium recruits those ER structures into Liberibacter containing vacuoles (LCVs), in which bacterial cells seem to propagate. ER- associated LCV formation was unique to CLas, as we could not detect these bodies in B. trigonica infected with CLso. ER recruitment is hypothesized to generate a safe replicative body to escape cellular immune responses in the insect gut. Understanding the molecular interactions that undelay these responses will open new opportunities for controlling CLas.

Pathogen vacuole purification from legionella-infected amoeba and macrophages.

PubMed

Hoffmann, Christine; Finsel, Ivo; Hilbi, Hubert

2013-01-01

Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.

Autophagy-related direct membrane import from ER/cytoplasm into the vacuole or apoplast: a hidden gateway also for secondary metabolites and phytohormones?

PubMed

Kulich, Ivan; Žárský, Viktor

2014-04-29

Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER) to vacuole (and also, apoplast) transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA) acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast), exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones' (e.g., auxin, abscisic acid (ABA) and salicylic acid (SA)) degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters) will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

PubMed Central

Kulich, Ivan; Žárský, Viktor

2014-01-01

Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER) to vacuole (and also, apoplast) transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA) acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast), exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA) and salicylic acid (SA)) degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters) will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging. PMID:24786101

Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[W

PubMed Central

Beauvoit, Bertrand P.; Colombié, Sophie; Monier, Antoine; Andrieu, Marie-Hélène; Biais, Benoit; Bénard, Camille; Chéniclet, Catherine; Dieuaide-Noubhani, Martine; Nazaret, Christine; Mazat, Jean-Pierre; Gibon, Yves

2014-01-01

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division. PMID:25139005

New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM).

PubMed

de Souza, Wanderley; Attias, Marcia

2015-07-01

The Helium Ion Microscope (HIM) is a new technology that uses a highly focused helium ion beam to scan and interact with the sample, which is not coated. The images have resolution and depth of field superior to field emission scanning electron microscopes. In this paper, we used HIM to study LLC-MK2 cells infected with Toxoplasma gondii. These samples were chemically fixed and, after critical point drying, were scraped with adhesive tape to expose the inner structure of the cell and parasitophorous vacuoles. We confirmed some of the previous findings made by field emission-scanning electron microscopy and showed that the surface of the parasite is rich in structures suggestive of secretion, that the nanotubules of the intravacuolar network (IVN) are not always straight, and that bifurcations are less frequent than previously thought. Fusion of the tubules with the parasite membrane or the parasitophorous vacuole membrane (PVM) was also infrequent. Tiny adhesive links were observed for the first time connecting the IVN tubules. The PVM showed openings of various sizes that even allowed the observation of endoplasmic reticulum membranes in the cytoplasm of the host cell. These findings are discussed in relation to current knowledge on the cell biology of T. gondii. Copyright © 2015 Elsevier Inc. All rights reserved.

Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamoto, C.; Matozaki, T.; Nagao, M.

1987-09-01

Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate (Gpp(NH)p)>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg/sup 2 +/. When pancreatic acini were treated withmore » 1 ..mu..g/ml pertussis toxin for 4 h, subsequent /sup 125/I-(Tyr/sup 1/)somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor.« less

Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

PubMed

Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro

2013-01-01

It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg') and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

Sudden Collapse of Vacuoles in Saintpaulia sp. Palisade Cells Induced by a Rapid Temperature Decrease

PubMed Central

Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro

2013-01-01

It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. ‘Iceberg’) and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury. PMID:23451194

High mobility group box 1 induces the activation of the Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway in pancreatic acinar cells in rats, while AG490 and rapamycin inhibit their activation.

PubMed

Wang, Guoliang; Zhang, Jingchao; Dui, Danhua; Ren, Haoyuan; Liu, Jin

2016-11-10

The pathogenesis of severe acute pancreatitis (SAP) remains unclear. The Janus kinase and signal transducer and activator of transcription (JAK/STAT) pathway is important for various cytokines and growth factors. This study investigated the effect of the late inflammatory factor high mobility group box 1 (HMGB1) on the activation of JAK2/STAT3 in pancreatic acinar cells and the inhibitory effects of AG490 (a JAK2 inhibitor) and rapamycin (a STAT3 inhibitor) on this pathway. Rat pancreatic acinar cells were randomly divided into the control, HMGB1, AG490, and rapamycin groups. The mRNA levels of JAK2 and STAT3 at 10, 30, 60, and 120 minutes were detected using reverse transcription polymerase chain reaction (RT-PCR). The protein levels of JAK2 and STAT3 at 60 and 120 minutes were observed using Western blotting. Compared with the control group, the HMGB1 group exhibited significantly increased levels of JAK2 mRNA at each time point; STAT3 mRNA at 30, 60, and 120 minutes; and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). Compared with the HMGB1 group, the AG490 and rapamycin groups both exhibited significantly decreased levels of JAK2 mRNA at each time point (p < 0.05); STAT3 mRNA at 30, 60, and 120 minutes (p < 0.01); and JAK2 and STAT3 proteins at 60 and 120 minutes (p < 0.01). HMGB1 induces the activation of the JAK2/STAT3 signaling pathway in rat pancreatic acinar cells, and this activation can be inhibited by AG490 and rapamycin. The results of this study may provide new insights for the treatment of SAP.

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

PubMed

Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

2013-03-04

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis

PubMed Central

Ellis, Kathryn; Bagwell, Jennifer

2013-01-01

The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H+-ATPase–dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development. PMID:23460678

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

PubMed Central

Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

2017-01-01

Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

PubMed

Lee, Yan Quan; Goh, Amanda S P; Ch'ng, Jun Hong; Nosten, François H; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S W

2014-01-01

Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium-driven bile uptake.

PubMed

Ferdek, Pawel E; Jakubowska, Monika A; Gerasimenko, Julia V; Gerasimenko, Oleg V; Petersen, Ole H

2016-11-01

Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas. Bile acids are known to induce Ca 2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored. Here we show that cholate and taurocholate elicit more dramatic Ca 2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3-sulfate primarily affects acinar cells. Ca 2+ signals and necrosis are strongly dependent on extracellular Ca 2+ as well as Na + ; and Na + -dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells. Bile acid-mediated pancreatic damage can be further escalated by bradykinin-induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca 2+ signals and necrosis in acinar cells. However, bile acid-elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca 2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3-sulfate (TLC-S), known to induce Ca 2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca 2+ signals on extracellular Na + and the presence of sodium-taurocholate cotransporting polypeptide (NTCP) indicate a Na + -dependent bile acid

Bile acids induce necrosis in pancreatic stellate cells dependent on calcium entry and sodium‐driven bile uptake

PubMed Central

Jakubowska, Monika A.; Gerasimenko, Julia V.; Gerasimenko, Oleg V.; Petersen, Ole H.

2016-01-01

Key points Acute biliary pancreatitis is a sudden and severe condition initiated by bile reflux into the pancreas.Bile acids are known to induce Ca2+ signals and necrosis in isolated pancreatic acinar cells but the effects of bile acids on stellate cells are unexplored.Here we show that cholate and taurocholate elicit more dramatic Ca2+ signals and necrosis in stellate cells compared to the adjacent acinar cells in pancreatic lobules; whereas taurolithocholic acid 3‐sulfate primarily affects acinar cells.Ca2+ signals and necrosis are strongly dependent on extracellular Ca2+ as well as Na+; and Na+‐dependent transport plays an important role in the overall bile acid uptake in pancreatic stellate cells.Bile acid‐mediated pancreatic damage can be further escalated by bradykinin‐induced signals in stellate cells and thus killing of stellate cells by bile acids might have important implications in acute biliary pancreatitis. Abstract Acute biliary pancreatitis, caused by bile reflux into the pancreas, is a serious condition characterised by premature activation of digestive enzymes within acinar cells, followed by necrosis and inflammation. Bile acids are known to induce pathological Ca2+ signals and necrosis in acinar cells. However, bile acid‐elicited signalling events in stellate cells remain unexplored. This is the first study to demonstrate the pathophysiological effects of bile acids on stellate cells in two experimental models: ex vivo (mouse pancreatic lobules) and in vitro (human cells). Sodium cholate and taurocholate induced cytosolic Ca2+ elevations in stellate cells, larger than those elicited simultaneously in the neighbouring acinar cells. In contrast, taurolithocholic acid 3‐sulfate (TLC‐S), known to induce Ca2+ oscillations in acinar cells, had only minor effects on stellate cells in lobules. The dependence of the Ca2+ signals on extracellular Na+ and the presence of sodium–taurocholate cotransporting polypeptide (NTCP) indicate a Na

A comprehensive computational human lung model incorporating inter-acinar dependencies: Application to spontaneous breathing and mechanical ventilation.

PubMed

Roth, Christian J; Ismail, Mahmoud; Yoshihara, Lena; Wall, Wolfgang A

2017-01-01

In this article, we propose a comprehensive computational model of the entire respiratory system, which allows simulating patient-specific lungs under different ventilation scenarios and provides a deeper insight into local straining and stressing of pulmonary acini. We include novel 0D inter-acinar linker elements to respect the interplay between neighboring alveoli, an essential feature especially in heterogeneously distended lungs. The model is applicable to healthy and diseased patient-specific lung geometries. Presented computations in this work are based on a patient-specific lung geometry obtained from computed tomography data and composed of 60,143 conducting airways, 30,072 acini, and 140,135 inter-acinar linkers. The conducting airways start at the trachea and end before the respiratory bronchioles. The acini are connected to the conducting airways via terminal airways and to each other via inter-acinar linkers forming a fully coupled anatomically based respiratory model. Presented numerical examples include simulation of breathing during a spirometry-like test, measurement of a quasi-static pressure-volume curve using a supersyringe maneuver, and volume-controlled mechanical ventilation. The simulations show that our model incorporating inter-acinar dependencies successfully reproduces physiological results in healthy and diseased states. Moreover, within these scenarios, a deeper insight into local pressure, volume, and flow rate distribution in the human lung is investigated and discussed. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse.

PubMed

Suto, Jun-Ichi

2012-11-06

Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F2 female mice: F2Ay mice (F2 mice with the Ay allele) and F2 non-Ay mice (F2 mice without the Ay allele). These were produced by crossing B6 females and DDD.Cg-Ay males. DDD.Cg-Ay is a congenic mouse strain for the Ay allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The Ay allele is dominant and homozygous lethal; therefore, living Ay mice are invariably heterozygotes. Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F2 non-Ay mice, and on chromosomes 2, 6, and 12 for F2Ay mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8. The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone. Identifying the genes responsible for the QTLs will be

Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse

PubMed Central

2012-01-01

Background Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F2 female mice: F2Ay mice (F2 mice with the Ay allele) and F2 non-Ay mice (F2 mice without the Ay allele). These were produced by crossing B6 females and DDD.Cg-Ay males. DDD.Cg-Ay is a congenic mouse strain for the Ay allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The Ay allele is dominant and homozygous lethal; therefore, living Ay mice are invariably heterozygotes. Results Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F2 non-Ay mice, and on chromosomes 2, 6, and 12 for F2Ay mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8. Conclusions The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone. Identifying the genes

Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles.

PubMed

Woo, Sang Su; James, Declan J; Martin, Thomas F J

2017-03-15

Munc13-4 is a Ca 2+ -dependent SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca 2+ -evoked secretion in various secretory cells. Studies in mast cell-like RBL-2H3 cells provide direct evidence that Munc13-4 with its two Ca 2+ -binding C2 domains functions as a Ca 2+ sensor for SG exocytosis. Unexpectedly, Ca 2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4 + /Rab7 + /Rab11 + endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4 + /Rab7 + SGs, followed by a merge with Rab11 + endosomes, and depended on Ca 2+ binding to Munc13-4. Munc13-4 promoted the Ca 2+ -stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca 2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. © 2017 Woo et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells.

PubMed

Chou, Ya-Shuan; Young, Tai-Horng; Lou, Pei-Jen

2015-11-01

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the

Restricted diffusion in a model acinar labyrinth by NMR: Theoretical and numerical results

NASA Astrophysics Data System (ADS)

Grebenkov, D. S.; Guillot, G.; Sapoval, B.

2007-01-01

A branched geometrical structure of the mammal lungs is known to be crucial for rapid access of oxygen to blood. But an important pulmonary disease like emphysema results in partial destruction of the alveolar tissue and enlargement of the distal airspaces, which may reduce the total oxygen transfer. This effect has been intensively studied during the last decade by MRI of hyperpolarized gases like helium-3. The relation between geometry and signal attenuation remained obscure due to a lack of realistic geometrical model of the acinar morphology. In this paper, we use Monte Carlo simulations of restricted diffusion in a realistic model acinus to compute the signal attenuation in a diffusion-weighted NMR experiment. We demonstrate that this technique should be sensitive to destruction of the branched structure: partial removal of the interalveolar tissue creates loops in the tree-like acinar architecture that enhance diffusive motion and the consequent signal attenuation. The role of the local geometry and related practical applications are discussed.

Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

PubMed

Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

2014-03-01

Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression. © 2013 John Wiley & Sons Ltd.

A multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis japonica.

PubMed

Takanashi, Kojiro; Yamada, Yasuyuki; Sasaki, Takayuki; Yamamoto, Yoko; Sato, Fumihiko; Yazaki, Kazufumi

2017-06-01

Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a multidrug and toxic compound extrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis japonica cells. CjMATE1 was found to localize at tonoplasts in C. japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1, a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara-Nishimura, I.; Nishimura, M.

1987-10-01

The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulse-labeled with (/sup 35/S)methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, ..gamma.. and delta. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin bymore » the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg/sup 2 +/, and Cu/sup 2 +/, but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles.« less

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

PubMed Central

Ko, Young-Joon; Lee, Miriam; Kang, KyeongJin; Song, Woo Keun; Jun, Youngsoo

2014-01-01

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms. PMID:24821814

Effects of muscarinic, alpha-adrenergic, and substance P agonists and ionomycin on ion transport mechanisms in the rat parotid acinar cell. The dependence of ion transport on intracellular calcium

PubMed Central

1989-01-01

The relationship between receptor-mediated increases in the intracellular free calcium concentration [( Ca]i) and the stimulation of ion fluxes involved in fluid secretion was examined in the rat parotid acinar cell. Agonist-induced increases in [Ca]i caused the rapid net loss of up to 50-60% of the total content of intracellular chloride (Cli) and potassium (Ki), which is consistent with the activation of calcium-sensitive chloride and potassium channels. These ion movements were accompanied by a 25% reduction in the intracellular volume. The relative magnitudes of the losses of Ki and the net potassium fluxes promoted by carbachol (a muscarinic agonist), phenylephrine (an alpha-adrenergic agonist), and substance P were very similar to their characteristic effects on elevating [Ca]i. Carbachol stimulated the loss of Ki through multiple efflux pathways, including the large-conductance Ca-activated K channel. Carbachol and substance P increased the levels of intracellular sodium (Nai) to more than 2.5 times the normal level by stimulating the net uptake of sodium through multiple pathways; Na-K-2Cl cotransport accounted for greater than 50% of the influx, and approximately 20% was via Na-H exchange, which led to a net alkalinization of the cells. Ionomycin stimulated similar fluxes through these two pathways, but also promoted sodium influx through an additional pathway which was nearly equivalent in magnitude to the combined uptake through the other two pathways. The carbachol- induced increase in Nai and decrease in Ki stimulated the activity of the sodium pump, measured by the ouabain-sensitive rate of oxygen consumption, to nearly maximal levels. In the absence of extracellular calcium or in cells loaded with the calcium chelator BAPTA (bis[o- aminophenoxy]ethane-N,N,N',N'-tetraacetic acid) the magnitudes of agonist- or ionomycin-stimulated ion fluxes were greatly reduced. The parotid cells displayed a marked desensitization to substance P; within 10 min the

TP53 alterations in pancreatic acinar cell carcinoma: new insights into the molecular pathology of this rare cancer.

PubMed

La Rosa, Stefano; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Furlan, Daniela; Sahnane, Nora; Vanoli, Alessandro; Albarello, Luca; Zhang, Lizhi; Notohara, Kenji; Casnedi, Selenia; Chenard, Marie-Pierre; Adsay, Volkan; Asioli, Sofia; Capella, Carlo; Sessa, Fausto

2016-03-01

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) are poorly understood and have been reported as being different from those in ductal adenocarcinomas. Loss of TP53 gene function in the pathogenesis of ACCs is controversial since contradictory findings have been published. A comprehensive analysis of the different possible genetic and epigenetic mechanisms leading to TP53 alteration in ACC has never been reported and hence the role of TP53 in the pathogenesis and/or progression of ACC remains unclear. We investigated TP53 alterations in 54 tumor samples from 44 patients, including primary and metastatic ACC, using sequencing analysis, methylation-specific multiplex ligation probe amplification, fluorescence in situ hybridization, and immunohistochemistry. TP53 mutations were found in 13 % of primary ACCs and in 31 % of metastases. Primary ACCs and metastases showed the same mutational profile, with the exception of one case, characterized by a wild-type sequence in the primary carcinoma and a mutation in the corresponding metastasis. FISH analysis revealed deletion of the TP53 region in 53 % of primary ACCs and in 50 % of metastases. Promoter hypermethylation was found in one case. The molecular alterations correlated well with the immunohistochemical findings. A statistically significant association was found between the combination of mutation of one allele and loss of the other allele of TP53 and worse survival.

Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones.

PubMed

Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

2015-01-01

The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production.

Jaspine B induces nonapoptotic cell death in gastric cancer cells independently of its inhibition of ceramide synthase.

PubMed

Cingolani, Francesca; Simbari, Fabio; Abad, Jose Luis; Casasampere, Mireia; Fabrias, Gemma; Futerman, Anthony H; Casas, Josefina

2017-08-01

Sphingolipids (SLs) have been extensively investigated in biomedical research due to their role as bioactive molecules in cells. Here, we describe the effect of a SL analog, jaspine B (JB), a cyclic anhydrophytosphingosine found in marine sponges, on the gastric cancer cell line, HGC-27. JB induced alterations in the sphingolipidome, mainly the accumulation of dihydrosphingosine, sphingosine, and their phosphorylated forms due to inhibition of ceramide synthases. Moreover, JB provoked atypical cell death in HGC-27 cells, characterized by the formation of cytoplasmic vacuoles in a time and dose-dependent manner. Vacuoles appeared to originate from macropinocytosis and triggered cytoplasmic disruption. The pan-caspase inhibitor, z-VAD, did not alter either cytotoxicity or vacuole formation, suggesting that JB activates a caspase-independent cell death mechanism. The autophagy inhibitor, wortmannin, did not decrease JB-stimulated LC3-II accumulation. In addition, cell vacuolation induced by JB was characterized by single-membrane vacuoles, which are different from double-membrane autophagosomes. These findings suggest that JB-induced cell vacuolation is not related to autophagy and it is also independent of its action on SL metabolism. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Leucine Affects α-Amylase Synthesis through PI3K/Akt-mTOR Signaling Pathways in Pancreatic Acinar Cells of Dairy Calves.

PubMed

Guo, Long; Liang, Ziqi; Zheng, Chen; Liu, Baolong; Yin, Qingyan; Cao, Yangchun; Yao, Junhu

2018-05-23

Dietary nutrient utilization, particularly starch, is potentially limited by digestion in dairy cow small intestine because of shortage of α-amylase. Leucine acts as an effective signal molecular in the mTOR signaling pathway, which regulates a series of biological processes, especially protein synthesis. It has been reported that leucine could affect α-amylase synthesis and secretion in ruminant pancreas, but mechanisms have not been elaborated. In this study, pancreatic acinar (PA) cells were used as a model to determine the cellular signal of leucine influence on α-amylase synthesis. PA cells were isolated from newborn Holstein dairy bull calves and cultured in Dulbecco's modifed Eagle's medium/nutrient mixture F12 liquid media containing four leucine treatments (0, 0.23, 0.45, and 0.90 mM, respectively), following α-amylase activity, zymogen granule, and signal pathway factor expression detection. Rapamycin, a specific inhibitor of mTOR, was also applied to PA cells. Results showed that leucine increased ( p < 0.05) synthesis of α-amylase as well as phosphorylation of PI3K, Akt, mTOR, and S6K1 while reduced ( p < 0.05) GCN2 expression. Inhibition of mTOR signaling downregulated the α-amylase synthesis. In addition, the extracellular leucine dosage significantly influenced intracellular metabolism of isoleucine ( p < 0.05). Overall, leucine regulates α-amylase synthesis through promoting the PI3K/Akt-mTOR pathway and reducing the GCN2 pathway in PA cells of dairy calves. These pathways form the signaling network that controls the protein synthesis and metabolism. It would be of great interest in future studies to explore the function of leucine in ruminant nutrition.

Autophagic Vacuolation Induced by Excess ROS Generation in HABP1/p32/gC1qR Overexpressing Fibroblasts and Its Reversal by Polymeric Hyaluronan

PubMed Central

Saha, Paramita; Chowdhury, Anindya Roy; Dutta, Shubhra; Chatterjee, Soumya; Ghosh, Ilora; Datta, Kasturi

2013-01-01

The ubiquitous hyaladherin, hyaluronan-binding protein 1 (HABP1/p32/gC1qR) upon stable overexpression in normal fibroblasts (F-HABP07) has been reported to induce mitochondrial dysfunction, growth retardation and apoptosis after 72 h of growth. HABP1 has been observed to accumulate in the mitochondria resulting in generation of excess Reactive Oxygen Species (ROS), mitochondrial Ca++ efflux and drop in mitochondrial membrane potential. In the present study, autophagic vacuolation was detected with monodansylcadaverin (MDC) staining from 36 h to 60 h of culture period along with elevated level of ROS in F-HABP07 cells. Increased expression of autophagic markers like MAP-LC3-II, Beclin 1 and autophagic modulator, DRAM confirmed the occurrence of the phenomenon. Reduced vacuole formation was observed upon treatment with 3-MA, a known PI3 kinase inhibitor, only at 32 h and was ineffective if treated later, as high ROS level was already attained. Treatment of F111 and F-HABP07 cells with bafilomycin A1 further indicated an increase in autophagosome formation along with autophagic degradation in HABP1 overexpressed fibroblasts. Comparison between normal fibroblast (F111) and F-HABP07 cells indicate reduced level of polymeric HA, its depolymerization and perturbed HA-HABP1 interaction in F-HABP07. Interestingly, supplementation of polymeric HA, an endogenous ROS scavenger, in the culture medium prompted reduction in number of vacuoles in F-HABP07 along with drop in ROS level, implying that excess ROS generation triggers initiation of autophagic vacuole formation prior to apoptosis due to overexpression of HABP1. Thus, the phenomenon of autophagy takes place prior to apoptosis induction in the HABP1 overexpressing cell line, F-HABP07. PMID:24205125

ENDOCYTOSIS IN CHANG LIVER CELLS

PubMed Central

Wagner, Roger; Rosenberg, Murray; Estensen, Richard

1971-01-01

The addition of 0.08 M sucrose to a culture medium containing Chang-strain human liver cells causes intense cytoplasmic vacuolation. Electron microscopy of these cells grown inferritin, time-lapse cinematography, and radioautography reveal that the vacuoles arise by endocytosis and that the sucrose is taken into the cell and localized in the vacuoles. Tracer studies demonstrate that sucrose-3H provides a marker for quantitation of endocytosis and that it neither induces nor stimulates endocytosis. Electron micrographs of vacuolated liver cells show microfilaments in close proximity to the inside of the plasma membrane, in the pseudopodia, and to the cytoplasmic side of the membrane surrounding endocytosis vacuoles. Cytochalasin B (CB), a mold metabolite that inhibits various types of cell motility, has a dose-dependent inhibitory effect on the uptake of sucrose-3H by these cells. This inhibition is accompanied by a cessation of the movement of ruffles and pseudopodia on the surface of the cells and the formation of blebs which arise from the cell's surface. These morphological changes are quickly reversible upon removal of CB. Alterations in the appearance and location of microfilaments are also observed in CB-treated cells. PMID:4329157

The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

NASA Astrophysics Data System (ADS)

Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

2014-07-01

Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

PubMed Central

Hanson, Kirsten K.; Ressurreição, Ana S.; Buchholz, Kathrin; Prudêncio, Miguel; Herman-Ornelas, Jonathan D.; Rebelo, Maria; Beatty, Wandy L.; Wirth, Dyann F.; Hänscheid, Thomas; Moreira, Rui; Marti, Matthias; Mota, Maria M.

2013-01-01

Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials. PMID:23836641

Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones

PubMed Central

Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

2015-01-01

The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA

PubMed Central

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells

DIE-RNA: A Reproducible Strategy for the Digestion of Normal and Injured Pancreas, Isolation of Pancreatic Cells from Genetically Engineered Mouse Models and Extraction of High Quality RNA.

PubMed

Assi, Mohamad; Dauguet, Nicolas; Jacquemin, Patrick

2018-01-01

The isolation of ribonucleic acid (RNA) suitable for gene expression studies is challenging in the pancreas, due to its high ribonuclease activity. This is even more complicated during pancreatitis, a condition associated with inflammation and fibrosis. Our aim was to implement a time-effective and reproducible protocol to isolate high quality RNA from specific pancreatic cell subtypes, in normal and inflammatory conditions. We used two genetically engineered mouse models (GEMM), Ela-CreER/YFP and Sox9-CreER/YFP, to isolate acinar and ductal cells, respectively. To induce pancreatitis, mice received a caerulein treatment (125 μg/kg) for 8 and 72 h. We alternatively used EGTA and calcium buffers that contain collagenase P (0.6 mg/mL) to rapidly digest the pancreas into individual cells. Most of the cells from normal and injured pancreas were single-dissociated, exhibited a round morphology and did not incorporate trypan blue dye. Cell suspensions from Ela- and Sox9-CreER/YFP pancreas were then sorted by flow cytometry to isolate the YFP-positive acinar and ductal cells, respectively. Sorted cells kept a round shape and emitted fluorescence detected by the 38 HE green fluorescence filter. RNA was isolated by column-based purification approach. The RNA integrity number (RIN) was high in sorted acinar cell fractions treated with or without caerulein (8.6 ± 0.17 and 8.4 ± 0.09, respectively), compared to the whole pancreas fraction (4.8 ± 1.1). Given the low number of sorted ductal cells, the RIN value was slightly lower compared to acini (7.4 ± 0.4). Quantitative-PCR experiments indicated that sorted acinar and ductal cells express the specific acinar and ductal markers, respectively. Additionally, RNA preparations from caerulein-treated acinar cells were free from significant contamination with immune cell RNA. We thus validated the DIE (Digestion, Isolation, and Extraction)-RNA tool as a reproducible and efficient protocol to isolate pure acinar and ductal cells

Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

PubMed Central

White, W. H.; Johnson, D. I.

1997-01-01

Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance. PMID:9286667

MoVam7, a Conserved SNARE Involved in Vacuole Assembly, Is Required for Growth, Endocytosis, ROS Accumulation, and Pathogenesis of Magnaporthe oryzae

PubMed Central

Dou, Xianying; Wang, Qi; Qi, Zhongqiang; Song, Wenwen; Wang, Wei; Guo, Min; Zhang, Haifeng; Zhang, Zhengguang; Wang, Ping; Zheng, Xiaobo

2011-01-01

Soluble NSF attachment protein receptor (SNARE) proteins play a central role in membrane fusion and vesicle transport of eukaryotic organisms including fungi. We previously identified MoSce22 as a homolog of Saccharomyces cerevisiae SNARE protein Sec22 to be involved in growth, stress resistance, and pathogenicity of Magnaporthe oryzae. Here, we provide evidences that MoVam7, an ortholog of S. cerevisiae SNARE protein Vam7, exerts conserved functions in vacuolar morphogenesis and functions in pathogenicity of M. oryzae. Staining with neutral red and FM4-64 revealed the presence of abnormal fragmented vacuoles and an absence of the Spitzenkörper body in the ΔMovam7 mutant. The ΔMovam7 mutant also exhibited reduced vegetative growth, poor conidiation, and failure to produce the infection structure appressorium. Additionally, treatments with cell wall perturbing agents indicated weakened cell walls and altered distributions of the cell wall component chitin. Furthermore, the ΔMovam7 mutant showed a reduced accumulation of reactive oxygen species (ROS) in the hyphal apex and failed to cause diseases on the rice plant. In summary, our studies indicate that MoVam7, like MoSec22, is a component of the SNARE complex whose functions in vacuole assembly also underlies the growth, conidiation, appressorium formation, and pathogenicity of M. oryzae. Further studies of MoVam7, MoSec22, and additional members of the SNARE complex are likely to reveal critical mechanisms in vacuole formation and membrane trafficking that is linked to fungal pathogenicity. PMID:21283626

Probing the contractile vacuole as Achilles' heel of the biotrophic grapevine pathogen Plasmopara viticola.

PubMed

Tröster, Viktoria; Setzer, Tabea; Hirth, Thomas; Pecina, Anna; Kortekamp, Andreas; Nick, Peter

2017-09-01

The causative agent of Grapevine Downy Mildew, the oomycete Plasmopara viticola, poses a serious threat to viticulture. In the current work, the contractile vacuole of the zoospore is analysed as potential target for novel plant protection strategies. Using a combination of electron microscopy, spinning disc confocal microscopy, and video differential interference contrast microscopy, we have followed the genesis and dynamics of this vacuole required during the search for the stomata, when the non-walled zoospore is exposed to hypotonic conditions. This subcellular description was combined with a pharmacological study, where the functionality of the contractile vacuole was blocked by manipulation of actin, by Na, Cu, and Al ions or by inhibition of the NADPH oxidase. We further observe that RGD peptides (mimicking binding sites for integrins at the extracellular matrix) can inhibit the function of the contractile vacuole as well. Finally, we show that an extract from Chinese liquorice (Glycyrrhiza uralensis) proposed as biocontrol for Downy Mildews can efficiently induce zoospore burst and that this activity depends on the activity of NADPH oxidase. The effect of the extract can be phenocopied by its major compound, glycyrrhizin, suggesting a mode of action for this biologically safe alternative to copper products.

Effects of cevimeline on the immunolocalization of aquaporin-5 and the ultrastructure of salivary glands in Sjögren's syndrome model mice.

PubMed

Nishimura, Hidehiko; Yakeishi, Akira; Saga, Tsuyoshi; Yamaki, Koh-Ichi

2009-01-01

Sjögren's syndrome (SS) is an autoimmune disorder whose main symptoms include xerostomia and dry eyes. It has been demonstrated that abnormal expression of aquaporin (AQP)-5 in the parotid and submandibular glands in SS model mice was corrected by cevimeline. In the present study, we orally administered cevimeline hydrochloride (cevimeline) to female MRL/l mice, which are widely used as a model for SS, to immunohistochemically investigate the localization of AQP-5 in the salivary glands. We also assessed the ultrastructure of acinar cells in the submandibular glands. AQP-5 was expressed in the apical and lateral cell membranes of acinar cells in the parotid and submandibular glands of normal mice, but not in the sublingual glands. In contrast, AQP-5 was expressed not only in the cell membranes in the apical domains but also in the cytoplasm in the SS model mice, indicating that the localization of AQP-5 was disordered in the SS model mice. After administration of cevimeline, AQP-5 was predominantly localized in the cellular apical domains of the acinar cells. Electron microscopy revealed that administration of cevimeline to the SS model mice and normal mice markedly reduced the number of secretory granules, increased the area of the rough endoplasmic reticulum, and expanded the intercellular gaps in the cells of the submandibular acini. Condensed vacuoles were also observed in the Golgi apparatuses, indicating that secondary enhancement of secretion and production of saliva had occurred. Consequently, the results of the present study demonstrate that the administration of cevimeline to the SS model mice increased salivary secretion in the submandibular glands. Furthermore, cevimeline transiently normalized the localization of AQP-5 expression in the parotid and submandibular glands.

Epidermal growth factor inhibits rat pancreatic cell proliferation, causes acinar cell hypertrophy, and prevents caerulein-induced desensitization of amylase release.

PubMed

Morisset, J; Larose, L; Korc, M

1989-06-01

The in vivo effects of epidermal growth factor (EGF) on pancreatic growth and digestive enzyme concentrations were compared with the actions of the pancreatic secretagogue caerulein in the adult rat. EGF (10 micrograms/kg BW) did not alter pancreatic weight or protein content. However, this concentration of EGF inhibited [3H]thymidine incorporation into DNA by 44%, decreased DNA content by 20%, and increased the concentrations of amylase, chymotrypsinogen, and protein by 106%, 232%, and 42%, respectively. Pancreatic acini prepared from EGF-treated rats exhibited a characteristic secretory response to caerulein that was superimposable to that obtained in acini from saline-treated rats. In both groups of acini half-maximal and maximal stimulation of amylase release occurred at approximately 5 pM and 50 pM caerulein, respectively. In contrast to EGF, caerulein (1 microgram/kg BW) increased pancreatic weight by 29% and protein content by 59%, and enhanced [3H]thymidine incorporation into DNA by 70%. Although caerulein increased the concentrations of pancreatic amylase and chymotrypsinogen by 38% and 297%, respectively, pancreatic acini prepared from caerulein-treated rats were less sensitive to the actions of caerulein in vitro when compared with acini from control rats. Indeed, the EC50 was shift from 4.8 pM to 9.8 pM after 4 days of treatment. EGF potentiated the actions of caerulein on pancreatic weight, protein content, and chymotrypsinogen concentration, and prevented the caerulein-induced alteration in the secretory responsiveness of the acinar cell. Conversely, caerulein reversed the inhibitory effect of EGF on thymidine incorporation. These findings suggest that EGF may modulate the trophic effects of certain gastrointestinal hormones, and may participate in the regulation of pancreatic exocrine function in vivo.

The Chlamydial Inclusion Preferentially Intercepts Basolaterally Directed Sphingomyelin-Containing Exocytic Vacuoles

PubMed Central

Moore, Elizabeth R.; Fischer, Elizabeth R.; Mead, David J.; Hackstadt, Ted

2010-01-01

Chlamydiae replicate intracellularly within a unique vacuole termed the inclusion. The inclusion circumvents classical endosomal/lysosomal pathways but actively intercepts a subset of Golgi-derived exocytic vesicles containing sphingomyelin (SM) and cholesterol. To further examine this interaction, we developed a polarized epithelial cell model to study vectoral trafficking of lipids and proteins to the inclusion. We examined seven epithelial cell lines for their ability to form single monolayers of polarized cells and support chlamydial development. Of these cell lines, polarized colonic mucosal C2BBe1 cells were readily infected with Chlamydia trachomatis and remained polarized throughout infection. Trafficking of (6-((N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino)hexanoyl)sphingosine) (NBD-C6-ceramide) and its metabolic derivatives, NBD-glucosylceramide (GlcCer) and NBD-SM, was analyzed. SM was retained within L2-infected cells relative to mock-infected cells, correlating with a disruption of basolateral SM trafficking. There was no net retention of GlcCer within L2-infected cells and purification of C. trachomatis elementary bodies from polarized C2BBe1 cells confirmed that bacteria retained only SM. The chlamydial inclusion thus appears to preferentially intercept basolaterally-directed SM-containing exocytic vesicles, suggesting a divergence in SM and GlcCer trafficking. The observed changes in lipid trafficking were a chlamydia-specific effect because Coxiella burnetii-infected cells revealed no changes in GlcCer or SM polarized trafficking. PMID:18778406

Content and Vacuole/Extravacuole Distribution of Neutral Sugars, Free Amino Acids, and Anthocyanin in Protoplasts 1

PubMed Central

Wagner, George J.

1979-01-01

Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. γ-Methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents. Images PMID:16660921

Suspected natural lysosomal storage disease from ingestion of pink morning glory (Ipomoea carnea) in goats in northern Argentina.

PubMed

Ríos, Elvio E; Cholich, Luciana A; Chileski, Gabriela; García, Enrique N; Lértora, Javier; Gimeno, Eduardo J; Guidi, María G; Mussart, Norma; Teibler, Gladys P

2015-07-01

This study describes an occurrence of pink morning glory (Ipomoea carnea) intoxication in goats in northern Argentina. The clinical signs displayed by the affected animals were ataxia, lethargy, emaciation, hypertonia of the neck muscles, spastic paresis in the hind legs, abnormal postural reactions and death. The clinico-pathologic examination revealed that the affected animals were anemic and their serum level of aspartate aminotransferase was significantly increased. Cytoplasmic vacuolation in the Purkinje cells and pancreatic acinar cells was observed by histological examination. The neuronal lectin binding pattern showed a strong positive reaction to WGA (Triticum vulgaris), sWGA (succinylated T. vulgaris) and LCA (Lens culinaris). Although I. carnea is common in tropical regions, this is the first report of spontaneous poisoning in goats in Argentina.

Determinants of GBP Recruitment to Toxoplasma gondii Vacuoles and the Parasitic Factors That Control It

PubMed Central

Virreira Winter, Sebastian; Niedelman, Wendy; Jensen, Kirk D.; Rosowski, Emily E.; Julien, Lindsay; Spooner, Eric; Caradonna, Kacey; Burleigh, Barbara A.; Saeij, Jeroen P. J.; Ploegh, Hidde L.; Frickel, Eva-Maria

2011-01-01

IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs) are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1) upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV) correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii. PMID:21931713

Functional characterization of contractile vacuole isolated from Amoeba proteus.

PubMed

Nishihara, Eri; Shimmen, Teruo; Sonobe, Seiji

2004-12-01

Contractile vacuoles (CVs) released from cells of Amoeba proteus were used to analyze its function in vitro. When CV was transferred to a hypertonic medium, its volume decreased within 10 sec. When it was subsequently returned to its original medium, it quickly started swelling. However, it ruptured before recovering its initial volume. These results suggested that the CV membrane is semi-permeable and that the fluid is collected by the osmotic gradient in vivo. The water permeability of membrane of isolated CV was calculated from the rate of osmotic volume change to be 0.94 microm/sec . OsM. This high value suggested that CV membrane is equipped with water channel. CV contracted (or burst) quickly upon addition of 1 mM ATP. Contraction was induced by ATP, but not by other nucleotides, GTP, ITP, ADP, or the analogues of ATP, AMP-PNP and ATPgammaS. It was suggested that the contraction of isolated CV was caused by increase in the tension of its membrane by ATP.

Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant1[OPEN

PubMed Central

Mary, Viviane; Schnell Ramos, Magali; Gillet, Cynthia; Socha, Amanda L.; Giraudat, Jérôme; Agorio, Astrid; Merlot, Sylvain; Clairet, Colin; Kim, Sun A.; Punshon, Tracy; Guerinot, Mary Lou; Thomine, Sébastien

2015-01-01

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids. PMID:26232490

LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling

PubMed Central

2013-01-01

Introduction Lysyl oxidase-like 2 (LOXL2) is a matrix-remodeling enzyme that has been shown to play a key role in invasion and metastasis of breast carcinoma cells. However, very little is known about its role in normal tissue homeostasis. Here, we investigated the effects of LOXL2 expression in normal mammary epithelial cells to gain insight into how LOXL2 mediates cancer progression. Methods LOXL2 was expressed in MCF10A normal human mammary epithelial cells. The 3D acinar morphogenesis of these cells was assessed, as well as the ability of the cells to form branching structures on extracellular matrix (ECM)-coated surfaces. Transwell-invasion assays were used to assess the invasive properties of the cells. Clinically relevant inhibitors of ErbB2, lapatinib and Herceptin (traztuzumab), were used to investigate the role of ErbB2 signaling in this model. A retrospective study on a previously published breast cancer patient dataset was carried out by using Disease Specific Genomic Analysis (DSGA) to investigate the correlation of LOXL2 mRNA expression level with metastasis and survival of ErbB2-positive breast cancer patients. Results Fluorescence staining of the acini revealed increased proliferation, decreased apoptosis, and disrupted polarity, leading to abnormal lumen formation in response to LOXL2 expression in MCF10A cells. When plated onto ECM, the LOXL2-expressing cells formed branching structures and displayed increased invasion. We noted that LOXL2 induced ErbB2 activation through reactive oxygen species (ROS) production, and ErbB2 inhibition by using Herceptin or lapatinib abrogated the effects of LOXL2 on MCF10A cells. Finally, we found LOXL2 expression to be correlated with decreased overall survival and metastasis-free survival in breast cancer patients with ErbB2-positive tumors. Conclusions These findings suggest that LOXL2 expression in normal epithelial cells can induce abnormal changes that resemble oncogenic transformation and cancer progression

Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria.

PubMed

Kodama, Yuuki; Fujishima, Masahiro

2012-07-01

Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding. Copyright © 2011 Elsevier GmbH. All rights reserved.

Does prostate acinar adenocarcinoma with Gleason Score 3+3=6 have the potential to metastasize?

PubMed

Montironi, Rodolfo; Scarpelli, Marina; Mazzucchelli, Roberta; Lopez-Beltran, Antonio; Santoni, Matteo; Briganti, Alberto; Montorsi, Francesco; Cheng, Liang

2014-10-18

There is a worldwide debate involving clinicians, uropathologists as well as patients and their families on whether Gleason score 6 adenocarcinoma should be labelled as cancer. We report a case of man diagnosed with biopsy Gleason score 6 acinar adenocarcinoma and classified as low risk (based on a PSA of 5 ng/mL and stage cT2a) whose radical prostatectomy specimen initially showed organ confined Gleason score 3+3=6, WHO nuclear grade 3, acinar adenocarcinoma with lymphovascular invasion and secondary deposit in a periprostatic lymph node. When deeper sections were cut to the point that almost all the slice present in the paraffin block was sectioned, a small tumor area (<5% of the whole tumor) of Gleason pattern 4 (poorly formed glands) was found in an extraprostatic position. The epilogue was that the additional finding changed the final Gleason score to 3+3=6 with tertiary pattern 4 and the stage to pT3a. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_190.

Mixed acinar-neuroendocrine-ductal carcinoma of the pancreas: a tale of three lineages.

PubMed

Anderson, Mark J; Kwong, Christina A; Atieh, Mohammed; Pappas, Sam G

2016-06-02

Most pancreatic cancers arise from a single cell type, although mixed pancreatic carcinomas represent a rare exception. The rarity of these aggressive malignancies and the limitations of fine-needle aspiration (FNA) pose significant barriers to diagnosis and appropriate management. We report a case of a 54-year-old man presenting with abdominal pain, jaundice and a hypodense lesion within the uncinate process on CT. FNA suggested poorly differentiated adenocarcinoma, which was subsequently resected via pancreaticoduodenectomy. Pathological analysis yielded diagnosis of invasive mixed acinar-neuroendocrine-ductal pancreatic carcinoma. Given the rare and deadly nature of these tumours, clinicians must be aware of their pathophysiology and do practice with a high degree of clinical suspicion, when appropriate. Surgical resection and thorough pathological analysis with immunohistochemical staining and electron microscopy remain the standards of care for mixed pancreatic tumours without gross evidence of metastasis. Diligent characterisation of the presentation and histological findings associated with these neoplasms should continue in order to promote optimal diagnostic and therapeutic strategies. 2016 BMJ Publishing Group Ltd.

Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites.

PubMed

González Montoro, Ayelén; Auffarth, Kathrin; Hönscher, Carina; Bohnert, Maria; Becker, Thomas; Warscheid, Bettina; Reggiori, Fulvio; van der Laan, Martin; Fröhlich, Florian; Ungermann, Christian

2018-06-04

The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES. Copyright © 2018 Elsevier Inc. All rights reserved.

[Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus].

PubMed

Bagrov, Ia Iu; Manusova, N B

2011-01-01

Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.

Hitchhiking vesicular transport routes to the vacuole: Amyloid recruitment to the Insoluble Protein Deposit (IPOD)

PubMed Central

Kumar, Rajesh; Neuser, Nicole; Tyedmers, Jens

2017-01-01

ABSTRACT Sequestration of aggregates into specialized deposition sites occurs in many species across all kingdoms of life ranging from bacteria to mammals and is commonly believed to have a cytoprotective function. Yeast cells possess at least 3 different spatially separated deposition sites, one of which is termed “Insoluble Protein Deposit (IPOD)” and harbors amyloid aggregates. We have recently discovered that recruitment of amyloid aggregates to the IPOD uses an actin cable based recruitment machinery that also involves vesicular transport.1 Here we discuss how different proteins known to be involved in vesicular transport processes to the vacuole might act to guide amyloid aggregates to the IPOD. These factors include the Myosin V motor protein Myo2 involved in transporting vacuolar vesicles along actin cables, the transmembrane protein Atg9 involved in the recruitment of large precursor hydrolase complexes to the vacuole, the phosphatidylinositol/ phosphatidylcholine (PI/PC) transfer protein Sec 14 and the SNARE chaperone Sec 18. Furthermore, we present new data suggesting that the yeast dynamin homolog Vps1 is also crucial for faithful delivery of the amyloid model protein PrD-GFP to the IPOD. This is in agreement with a previously identified role for Vps1 in recruitment of heat-denatured aggregates to a perivacuolar deposition site.2 PMID:28277942

The vacuole model: new terms in the second order deflection of light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Amrita; Nandi, Kamal K.; Garipova, Guzel M.

2011-02-01

The present paper is an extension of a recent work (Bhattacharya et al. 2010) to the Einstein-Strauss vacuole model with a cosmological constant, where we work out the light deflection by considering perturbations up to order M{sup 3} and confirm the light bending obtained previously in their vacuole model by Ishak et al. (2008). We also obtain another local coupling term −5πM{sup 2}Λ/8 related to Λ, in addition to the one obtained by Sereno (2008, 2009). We argue that the vacuole method for light deflection is exclusively suited to cases where the cosmological constant Λ disappears from the path equation.more » However, the original Rindler-Ishak method (2007) still applies even if a certain parameter γ of Weyl gravity does not disappear. Here, using an alternative prescription, we obtain the known term −γR/2, as well as another new local term 3πγM/2 between M and γ. Physical implications are compared, where we argue that the repulsive term −γR/2 can be masked by the Schwarzschild term 2M/R in the halo regime supporting attractive property of the dark matter.« less

Role of leptin in modulation of Porphyromonas gingivalis lipopolysaccharide-induced up-regulation of endothelin-1 in salivary gland acinar cells.

PubMed

Slomiany, Bronislaw L; Slomiany, Amalia

2005-08-01

Leptin, a pleiotropic cytokine that regulates food intake and metabolic and endocrine responses, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection. In this study, we report that in sublingual salivary gland acinar cells leptin plays a role in the suppression of up-regulation in endothelin-1 (ET-1), induced by the LPS of a periodontopathic bacterium P. gingivalis. We show that P. gingivalisLPS detrimental effect on salivary mucin synthesis, associated with up-regulation (3.9-fold) in ET-1 generation and the enhancement (3.2-fold) in endothelin-converting enzyme-1 (ECE-1) activity, was subject to a dose-dependent suppression by leptin. The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, as well as by ERK inhibitor, PD98059. However, while the blockade of ERK led also to amplification in the impedance by leptin of the LPS-induced expression of ECE-1 and ET-1, the effect was not observed in the presence of wortmannin. The findings are the first to demonstrate that leptin counters the pathological consequences of P. gingivalisinfection on the synthesis of salivary mucin through the involvement in signaling events of PI3K and ERK pathways. We also show that the ERK cascade represents a critical signaling target for leptin in the LPS-induced up-regulation in ET-1.

Increasing cyclic electron flow is related to Na+ sequestration into vacuoles for salt tolerance in soybean.

PubMed

He, Yi; Fu, Junliang; Yu, Chenliang; Wang, Xiaoman; Jiang, Qinsu; Hong, Jian; Lu, Kaixing; Xue, Gangping; Yan, Chengqi; James, Andrew; Xu, Ligen; Chen, Jianping; Jiang, Dean

2015-11-01

In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Krüppel-like Factor 5, Increased in Pancreatic Ductal Adenocarcinoma, Promotes Proliferation, Acinar-to-Ductal Metaplasia, Pancreatic Intraepithelial Neoplasia, and Tumor Growth in Mice.

PubMed

He, Ping; Yang, Jong Won; Yang, Vincent W; Bialkowska, Agnieszka B

2018-04-01

Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar-to-ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs. We performed studies in adult mice with conditional disruption of Klf5 (Klf5 fl/fl ) and/or expression of Kras G12D (LSL-Kras G12D ) via Cre ERTM recombinase regulated by an acinar cell-specific promoter (Ptf1a). Activation of Kras G12D and loss of KLF5 was achieved by administration of tamoxifen. Pancreatitis was induced in mice by administration of cerulein; pancreatic tissues were collected, analyzed by histology and immunohistochemistry, and transcriptomes were compared between mice that did or did not express KLF5. We performed immunohistochemical analyses of human tissue microarrays, comparing levels of KLF5 among 96 human samples of PDAC. UN-KC-6141 cells (pancreatic cancer cells derived from Pdx1-Cre;LSL-Kras G12D mice) were incubated with inhibitors of different kinases and analyzed in proliferation assays and by immunoblots. Expression of KLF5 was knocked down with small hairpin RNAs or CRISPR/Cas9 strategies; cells were analyzed in proliferation and gene expression assays, and compared with cells expressing control vectors. Cells were subcutaneously injected into flanks of syngeneic mice and tumor growth was assessed. Of the 96 PDAC samples analyzed, 73% were positive for KLF5 (defined as nuclear staining in more than 5% of tumor cells). Pancreata from Ptf1a-Cre ERTM ;LSL-Kras G12D mice contained ADM and PanIN lesions, which contained high levels of nuclear KLF5 within these structures. In contrast, Ptf1a-Cre ERTM ;LSL-Kras G12D ;Klf5 fl

The Ethanolamine Permease EutH Promotes Vacuole Adaptation of Salmonella enterica and Listeria monocytogenes during Macrophage Infection.

PubMed

Anderson, Christopher J; Satkovich, John; Köseoğlu, Volkan K; Agaisse, Hervé; Kendall, Melissa M

2018-05-01

Ethanolamine is a ubiquitous and essential molecule within a host. Significantly, bacterial pathogens exploit ethanolamine during infection to promote growth and regulate virulence. The ethanolamine permease EutH is dispensable for growth in vitro under standard conditions, whereas EutH is required for ethanolamine utilization at low pH. These findings suggested a model in which EutH facilitates diffusion of ethanolamine into the bacterial cell in acidic environments. To date, the ecological significance of this model has not been thoroughly investigated, and the importance of EutH to bacterial growth under physiologically relevant conditions is not known. During infection, immune cells internalize invading bacteria within an acidic, nutrient-depleted vacuole called the phagosome. Here, we investigated the hypothesis that EutH promotes bacterial survival following phagocytosis. Our findings indicate that EutH is important for survival and replication of the facultative intracellular pathogens Salmonella enterica serovar Typhimurium and Listeria monocytogenes during prolonged or transient exposure to the phagosome, respectively. Furthermore, in agreement with EutH being important in the acidic environment, neutralization of the vacuole abolished the requirement for EutH. Significantly, consistent with a role for EutH in promoting intramacrophage survival, EutH was not required during S Typhimurium local intestinal infection but specifically conferred an advantage upon dissemination to peripheral organs. These findings reveal a physiologically relevant and conserved role for EutH in spatiotemporal niche adaptation during infection. Copyright © 2018 American Society for Microbiology.

Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome.

PubMed

Collins, H L; Schaible, U E; Ernst, J D; Russell, D G

1997-01-01

The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells.

Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

PubMed

Ploemen, Ivo H J; Croes, Huib J; van Gemert, Geert-Jan J; Wijers-Rouw, Mietske; Hermsen, Cornelus C; Sauerwein, Robert W

2012-01-01

The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

Smad2/3 Linker Phosphorylation Is a Possible Marker of Pancreatic Stem/Progenitor Cells in the Regenerative Phase of Acute Pancreatitis.

PubMed

Sakao, Masayuki; Sakaguchi, Yutaku; Suzuki, Ryo; Takahashi, Yu; Kishimoto, Masanobu; Fukui, Toshiro; Uchida, Kazushige; Nishio, Akiyoshi; Matsuzaki, Koichi; Okazaki, Kazuichi

The aims of this study are to characterize cell proliferation and differentiation during regeneration after pancreatitis and pancreatic buds during development to evaluate the role of Smad2/3, phosphorylated at the specific linker threonine residues (pSmad2/3L-Thr) in positive cells. Male C57BL/6 mice received hourly intraperitoneal injections of cerulein and were analyzed after induced pancreatitis. Pancreatitis-affected tissue sections and pancreatic buds were immunostained for pSmad2/3L-Thr, with other markers thought to be stem/progenitor markers of the pancreas. pSmad2/3L-Thr immunostaining-positive cells increased as the pancreatitis progressed. The expression of pSmad2/3L-Thr was seen in acinar cells and ductlike tubular complexes. These results suggest that pSmad2/3L-Thr is expressed during acinar-ductal metaplasia. Immunohistochemical colocalization of pSmad2/3L-Thr with Ki67 was never observed. pSmad2/3L-Thr-positive cells may remain in an undifferentiated state. During the pancreatic development process, pSmad2/3L-Thr was expressed as other markers. pSmad2/3L-Thr develops in duct structure of the undifferentiated cell population in the last part of viviparity that acinar structure is formed clearly. pSmad2/3L-Thr expression occurs during acinar-ductal metaplasia after pancreatitis and may represent the contribution of stem cells and/or progenitor cells to the differentiation of the pancreas.

Cell cycle-dependent induction of autophagy, mitophagy and reticulophagy.

PubMed

Tasdemir, Ezgi; Maiuri, M Chiara; Tajeddine, Nicolas; Vitale, Ilio; Criollo, Alfredo; Vicencio, José Miguel; Hickman, John A; Geneste, Olivier; Kroemer, Guido

2007-09-15

When added to cells, a variety of autophagy inducers that operate through distinct mechanisms and target different organelles for autophagic destruction (mitochondria in mitophagy, endoplasmic reticulum in reticulophagy) rarely induce autophagic vacuolization in more than 50% or the cells. Here we show that this heterogeneity may be explained by cell cycle-specific effects. The BH3 mimetic ABT737, lithium, rapamycin, tunicamycin or nutrient depletion stereotypically induce autophagy preferentially in the G(1) and S phases of the cell cycle, as determined by simultaneous monitoring of cell cycle markers and the cytoplasmic aggregation of GFP-LC3 in autophagic vacuoles. These results point to a hitherto neglected crosstalk between autophagic vacuolization and cell cycle regulation.

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PubMed

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

PubMed Central

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells.

PubMed

Gründling, Angelika; Gonzalez, Mark D; Higgins, Darren E

2003-11-01

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in

Late steps of egg cell differentiation are accelerated by pollination in Zea mays L.

PubMed

Mól, R; Idzikowska, K; Dumas, C; Matthys-Rochon, E

2000-04-01

Egg cells were analysed cytologically during the female receptivity period in maize (Zea mays L., line A 188). Three classes of egg cell were distinguished: type A--small, non-vacuolated cells with a central nucleus; type B--larger cells with small vacuoles surrounding the perinuclear cytoplasm located in the middle of the cell; type C--big cells with a large apical vacuole and the mid-basal perinuclear cytoplasm. The less-dense cytoplasm of the vacuolated egg cells usually contained numerous cup- or bell-shaped mitochondria. The three egg types appear to correspond to three late stages of egg cell differentiation. The frequencies of each of the three egg types were monitored in developing maize ears before and after pollination. In young ears, with the silks just extending out of the husks, small A-type cells were found in about 86% of ovules. Their frequency decreased to about 58% at the optimum silk length, remained unchanged in non-pollinated ears, and fell to 16% at the end of the female receptivity period. However, after pollination and before fertilisation the frequency of these cells decreased to about 33%, and the larger vacuolated egg cells (types B and C) prevailed. At various stages of the receptivity period, pollination accelerated changes in the egg population, increasing the number of ovules bearing larger, vacuolated egg cells. Experiments with silk removal demonstrated that putative pollination signals act immediately after pollen deposition and are not species-specific.

Bacterial spread from cell to cell: beyond actin-based motility.

PubMed

Kuehl, Carole J; Dragoi, Ana-Maria; Talman, Arthur; Agaisse, Hervé

2015-09-01

Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vieira da Silva, Claudio; Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sao Paulo, Rua Botucatu, 862, 6o andar, 04023-062 Sao Paulo, SP; Alves da Silva, Erika

2009-01-16

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RHmore » strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP{sub 2} and PIP{sub 3} to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.« less

Ylpex5 mutation partially suppresses the defective hyphal growth of a Yarrowia lipolytica ceramide synthase mutant, Yllac1, by recovering lipid raft polarization and vacuole morphogenesis.

PubMed

Bal, Jyotiranjan; Lee, Hye-Jeong; Cheon, Seon Ah; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Jeong-Yoon

2013-01-01

Sphingolipids are involved in cell differentiation and morphogenesis in eukaryotic cells. In this study, YlLac1p, a ceramide synthase required for glucosylceramide (GlcCer) synthesis, was found to be essential for hyphal growth in Yarrowia lipolytica. Y. lipolytica GlcCer was shown to be composed of a C16:0 fatty acid, which is hydroxylated at C2, and a C18:2 long chain base, which is unsaturated at both C4 and C8 and methylated at C9. Domain swapping analysis revealed that the entire TRAM/Lag1/CLN8 (TLC) domain, not the Lag1 motif, is crucial for the function of YlLac1p. YlDes1p, the C4 desaturase of the ceramide synthesized by YlLac1p, was also required for Y. lipolytica morphogenesis. Both Yllac1Δ and Yldes1Δ mutants neither polarize lipid rafts nor form normal vacuoles. Interestingly, mutation in YlPEX5, which encode a peroxisomal targeting signal receptor, partially suppressed the defective hyphal growth of Yllac1Δ. The Yllac1ΔYlpex5Δ mutant restored the ability to polarize lipid rafts and to form normal vacuoles, although it could not synthesize GlcCer. Taken together, our results suggest that GlcCer or GlcCer derivatives may be involved in hyphal morphogenesis in Y. lipolytica, at least in part, by affecting polarization of lipid rafts and vacuole morphogenesis. Copyright © 2012 Elsevier Inc. All rights reserved.

The quest for tissue stem cells in the pancreas and other organs, and their application in beta-cell replacement.

PubMed

Houbracken, Isabelle; Bouwens, Luc

2010-01-01

Adult stem cell research has drawn a lot of attention by many researchers, due to its medical hope of cell replacement or regenerative therapy for diabetes patients. Despite the many research efforts to date, there is no consensus on the existence of stem cells in adult pancreas. Genetic lineage tracing experiments have put into serious doubt whether β-cell neogenesis from stem/progenitor cells takes place postnatally. Different in vitro experiments have suggested centroacinar, ductal, acinar, stellate, or yet unidentified clonigenic cells as candidate β-cell progenitors. As in the rest of the adult stem cell field, sound and promising observations have been made. However, these observations still need to be replicated. As an alternative to committed stem/progenitor cells in the pancreas, transdifferentiation or lineage reprogramming of exocrine acinar and endocrine α-cells may be used to generate new β-cells. At present, it is unclear which approach is most medically promising. This article highlights the progress being made in knowledge about tissue stem cells, their existence and availability for therapy in diabetes. Particular attention is given to the assessment of methods to verify the existence of tissue stem cells.

Active Transport of Exogenous S-Adenosylmethionine and Related Compounds into Cells and Vacuoles of Saccharomyces cerevisiae

PubMed Central

Nakamura, K. D.; Schlenk, F.

1974-01-01

Saccharomyces cerevisiae 4094-B (α, ade-2, ura-1) in potassium phosphate buffer with glucose under aerobic conditions took up (−)S-adenosyl-l-methionine from the medium in sufficient quantity to permit the demonstration of its accumulation in the vacuole by ultraviolet micrography. The same result was obtained with (±)S-adenosyl-l-methionine, (±)S-adenosyl-d-methionine, and (−)S-adenosyl-l-ethionine. The rate of uptake was slow with (−)S-adenosyl-S(n-propyl)-l-homocysteine and S-adenosyl-d-homocysteine. S-Adenosyl-l-homocysteine was assimilated rapidly, but intracellular degradation precluded accumulation and ultraviolet micrographic studies. The uptake of 5′-methyl-, 5′-ethyl-, 5′-n-propylthioadenosine, and 5′-dimethylsulfonium adenosine was minimal. Images PMID:4371757

Current Progress in Tonoplast Proteomics Reveals Insights into the Function of the Large Central Vacuole

PubMed Central

Trentmann, Oliver; Haferkamp, Ilka

2013-01-01

Vacuoles of plants fulfill various biologically important functions, like turgor generation and maintenance, detoxification, solute sequestration, or protein storage. Different types of plant vacuoles (lytic versus protein storage) are characterized by different functional properties apparently caused by a different composition/abundance and regulation of transport proteins in the surrounding membrane, the tonoplast. Proteome analyses allow the identification of vacuolar proteins and provide an informative basis for assigning observed transport processes to specific carriers or channels. This review summarizes techniques required for vacuolar proteome analyses, like e.g., isolation of the large central vacuole or tonoplast membrane purification. Moreover, an overview about diverse published vacuolar proteome studies is provided. It becomes evident that qualitative proteomes from different plant species represent just the tip of the iceberg. During the past few years, mass spectrometry achieved immense improvement concerning its accuracy, sensitivity, and application. As a consequence, modern tonoplast proteome approaches are suited for detecting alterations in membrane protein abundance in response to changing environmental/physiological conditions and help to clarify the regulation of tonoplast transport processes. PMID:23459586

Effects of cardiac oscillations and lung volume on acinar gas mixing during apnea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mackenzie, C.F.; Skacel, M.; Barnas, G.M.

1990-05-01

We evaluated the importance of cardiogenic gas mixing in the acini of 13 dogs during 2 min of apnea. 133Xe (1-2 mCi in 4 ml of saline) was injected into an alveolar region through an occluded pulmonary artery branch, and washout was measured by gamma scintillation scanning during continued occlusion or with blood flow reinstated. The monoexponential rate constant for Xe washout (XeW) was -0.4 +/- 0.08 (SE) min-1 at functional residual capacity (FRC) with no blood flow in the injected region. It decreased by more than half at lung volumes 500 ml above and 392 ml below FRC. Withmore » intact pulmonary blood flow, XeW was -1.0 +/- 0.08 (SE) min-1 at FRC, and it increased with decreasing lung volume. However, if calculated Xe uptake by the blood was subtracted from the XeW measured with blood flow intact, resulting values at FRC and at FRC + 500 ml were not different from XeW with no blood flow. Reasonable calculation of Xe blood uptake at 392 ml below FRC was not possible because airway closure, increased shunt, and other factors affect XeW. After death, no significant XeW could be measured, which suggests that XeW caused by molecular diffusion was small. We conclude that (1) the effect of heart motion on the lung parenchyma increases acinar gas mixing during apnea, (2) this effect diminishes above or below FRC, and (3) there is probably no direct effect of pulmonary vascular pulsatility on acinar gas mixing.« less

APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation.

PubMed

Furlan, Daniela; Sahnane, Nora; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Marando, Alessandro; Zhang, Lizhi; Vanoli, Alessandro; Casnedi, Selenia; Adsay, Volkan; Notohara, Kenji; Albarello, Luca; Asioli, Sofia; Sessa, Fausto; Capella, Carlo; La Rosa, Stefano

2014-05-01

Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.

1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Asare, Nana; Landvik, Nina E.; Lagadic-Gossmann, Dominique

2008-07-15

Mechanistic studies of nitro-PAHs (polycyclic aromatic hydrocarbons) of interest might help elucidate which chemical characteristics are most important in eliciting toxic effects. 1-Nitropyrene (1-NP) is the predominant nitrated PAH emitted in diesel exhaust. 1-NP-exposed Hepa1c1c7 cells exhibited marked changes in cellular morphology, decreased proliferation and different forms of cell death. A dramatic increase in cytoplasmic vacuolization was observed already after 6 h of exposure and the cells started to round up at 12 h. The rate of cell proliferation was markedly reduced at 24 h and apoptotic as well as propidium iodide (PI)-positive cells appeared. Electron microscopic examination revealed thatmore » the vacuolization was partly due to mitochondria swelling. The caspase inhibitor Z-VAD-FMK inhibited only the apoptotic cell death and Nec-1 (an inhibitor of necroptosis) exhibited no inhibitory effects on either cell death or vacuolization. In contrast, cycloheximide markedly reduced both the number of apoptotic and PI-positive cells as well as the cytoplasmic vacuolization, suggesting that 1-NP induced paraptotic cell death. All the MAPKs; ERK1/2, p38 and JNK, appear to be involved in the death process since marked activation was observed upon 1-NP exposure, and their inhibitors partly reduced the induced cell death. The ERK1/2 inhibitor PD 98057 completely blocked the induced vacuolization, whereas the other MAPKs inhibitors only had minor effects on this process. These findings suggest that 1-NP may cause apoptosis and paraptosis. In contrast, the corresponding amine (1-aminopyrene) elicited only minor apoptotic and necrotic cell death, and cells with characteristics typical of paraptosis were absent.« less

Efficacy of Yeast' Vacuoles as Antimicrobial Agents to Escherichia coli Bacteremia in Rat.

PubMed

Yoon, Jihee; Cho, Ho-Seong; Park, Chul; Park, Byoung-Yong; Kim, Yang-Hoon; Min, Jiho

2017-01-01

Yeast vacuoles, lysosomes, are cell organelles that have antimicrobial activity against several bacteria in vitro. Lysosomes have a potential application to the treatment of pathogens such as antibiotics in vivo. Therefore, the in vivo efficacy of lysosomes was examined in a rat infection model against pathogenic Escherichia coli with varying susceptibilities to standard antimicrobial agents. Before in vivo testing, the concentration-dependent safety of lysosomes was confirmed by blood test and histopathology of normal rats. The therapeutic efficacy of lysosomes was examined in terms of the survival of E. coli in infected rat blood. The complete blood count and histopathology results were affected by the lysosomes concentration. In addition, the E. coli growth was inhibited by the initial injection of lysosomes. These results support the use of lysosomes as a bacterial inhibitor of an infected rat model.

New advances in cell physiology and pathophysiology of the exocrine pancreas.

PubMed

Mössner, Joachim

2010-01-01

This review provides some aspects on the physiology of stimulation and inhibition of pancreatic digestive enzyme secretion and the pathophysiology of pancreatic acinar cell function leading to pancreatitis. Cholecystokinin (CCK) stimulates both directly via CCK-A receptors on acinar cells and indirectly via CCK-B receptors on nerves, followed by acetylcholine release, pancreatic enzyme secretion. It is still not known whether CCK-A receptors exist in human acinar cells, in contrast to acinar cells of rodents where CCK-A receptors have been well described. CCK has numerous actions both in the periphery and in the central nervous systems. CCK inhibits gastric motility and regulates satiety. Another major function of CCK is stimulation of gallbladder contraction. This function enables that bile acids act simultaneously with pancreatic lipolytic enzymes. Secretin is a major stimulator of bicarbonate secretion. Trypsinogen is activated by the gut mucosal enzyme enterokinase. The other pancreatic proenzymes are activated by trypsin. Termination of enzyme secretion may be regulated by negative feedback mechanisms via destruction of CCK-releasing peptides by trypsin. Furthermore, the ileum may act as a brake by release of inhibitory hormones such as PYY and somatostatin. In the pathophysiology of acute pancreatitis, fusion of zymogen granules with lysosomes leading to intracellular activation of trypsinogen is regarded as an initiation step. This activation of trypsinogen may be caused by the lysosomal enzyme cathepsin B. However, autoactivation of trypsinogen itself may be a possibility in pathogenesis. Autoactivation is enhanced in certain mutations of trypsinogen. Furthermore, an imbalance of protease inhibitors and active proteases may be involved. The role of pancreatic lipolytic enzymes, the role of bicarbonate secretion, and toxic Ca(2+) signals by excessive liberation from the endoplasmic reticulum have to be discussed in the pathogenesis of acute pancreatitis

LegC3, an Effector Protein from Legionella pneumophila, Inhibits Homotypic Yeast Vacuole Fusion In Vivo and In Vitro

PubMed Central

Bennett, Terry L.; Kraft, Shannon M.; Reaves, Barbara J.; Mima, Joji; O’Brien, Kevin M.; Starai, Vincent J.

2013-01-01

During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes). This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis. PMID:23437241

Reconstructing human pancreatic differentiation by mapping specific cell populations during development.

PubMed

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-07-21

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2 + population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3 , a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated.

Listeriolysin O Membrane Damaging Activity Involves Arc Formation and Lineaction -- Implication for Listeria monocytogenes Escape from Phagocytic Vacuole

PubMed Central

Ruan, Yi; Rezelj, Saša; Bedina Zavec, Apolonija; Anderluh, Gregor; Scheuring, Simon

2016-01-01

Listeriolysin-O (LLO) plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood, probably due to unavailability of suitable experimental tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed atomic force microscopy (HS-AFM) featuring high spatio-temporal resolution on model membranes and optical microscopy on giant unilamellar vesicles (GUVs) to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concentration and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context. PMID:27104344

A case report of mixed acinar-endocrine carcinoma of the pancreas treated with S-1 chemotherapy: Does it work or induce endocrine differentiation?

PubMed

Yokode, Masataka; Itai, Ryosuke; Yamashita, Yukimasa; Zen, Yoh

2017-11-01

Acinar cell carcinomas (ACCs) and mixed acinar-endocrine carcinomas (MAECs) of the pancreas are rare, accounting for only 1% of pancreatic tumors. Although both typically present at an advanced stage, chemotherapeutic regimes have not yet been standardized. A 65-year-old man presented with a large mass in the pancreatic tail with multiple liver metastases. He was initially treated with gemcitabine for suspected ductal carcinoma of the pancreas, but no response was observed. S-1, administered as second-line chemotherapy, showed an approximately 38% reduction in the size of the primary tumor and metastatic deposits with therapeutic effects being maintained for 12 months. When the tumor progressed again, he underwent a percutaneous liver biopsy, which led to the diagnosis of MAEC. Combination therapy with cisplatin and etoposide targeting the endocrine component was administered, and this was based on the endocrine component potentially being less sensitive to S-1 than the ACC element. However, therapy was stopped due to the development of neutropenia, and the patient is currently receiving best supportive care. Given the previous studies suggested that S-1 is more effective for ACCs than gemcitabine, MAECs may also respond to S-1 chemotherapy, similar to ACCs. Another potential interpretation is that S-1 was effective when the condition was ACC, and eventually showed decreased effectiveness when the condition shifted to MAEC. Future studies are needed to conclude whether S-1 chemotherapy truly works against MAECs or induces endocrine differentiation in ACCs as a part of the drug-resistance process.

A fluorescent reporter protein containing AtRMR1 domains is targeted to the storage and central vacuoles in Arabidopsis thaliana and tobacco leaf cells.

PubMed

Scabone, Camila María; Frigerio, Lorenzo; Petruccelli, Silvana

2011-10-01

To develop a new strategy to target recombinant proteins to the vacuolar storage system in transgenic plants, the ability of the transmembrane and cytosolic domains of Arabidopsis receptor homology-transmembrane-RING H2-1 (AtRMR1) was evaluated. A secreted version of RFP (secRFP) and a fusion of it to the transmembrane and cytosolic domains of AtRMR1 (RFP-TMCT) were produced and studied both in transient and stable expression assays. Transient expression in leaves of Nicotiana tabacum showed that secRFP is secreted to the apoplast while its fusion to TMCT of AtRMR1 is sufficient to prevent secretion of the reporter. In tobacco leaves, RFP-TMCT reporter showed an endoplasmic reticulum pattern in early expression stages while in late expression stages, it was found in the vacuolar lumen. For the first time, the role of TM and CT domains of AtRMR1 in stable expression in Arabidopsis thaliana is presented; the fusion of TMCT to secRFP is sufficient to sort RFP to the lumen of the central vacuoles in leaves and roots and to the lumen of PSV in cotyledons of mature embryos. In addition, biochemical studies performed in extract from transgenic plants showed that RFP-TMCT is an integral membrane protein. Full-length RFP-TMCT was also found in the vacuolar lumen, suggesting internalization into destination vacuole. Not colocalization of RFP-TMCT with tonoplast and plasma membrane markers were observed. This membrane vacuolar determinant sorting signal could be used for future application in molecular pharming as an alternative means to sort proteins of interest to vacuoles.

Zanthoxylum fruit extract from Japanese pepper promotes autophagic cell death in cancer cells.

PubMed

Nozaki, Reo; Kono, Toru; Bochimoto, Hiroki; Watanabe, Tsuyoshi; Oketani, Kaori; Sakamaki, Yuichi; Okubo, Naoto; Nakagawa, Koji; Takeda, Hiroshi

2016-10-25

Zanthoxylum fruit, obtained from the Japanese pepper plant (Zanthoxylum piperitum De Candolle), and its extract (Zanthoxylum fruit extract, ZFE) have multiple physiological activities (e.g., antiviral activity). However, the potential anticancer activity of ZFE has not been fully examined. In this study, we investigated the ability of ZFE to induce autophagic cell death (ACD). ZFE caused remarkable autophagy-like cytoplasmic vacuolization, inhibited cell proliferation, and ultimately induced cell death in the human cancer cell lines DLD-1, HepG2, and Caco-2, but not in A549, MCF-7, or WiDr cells. ZFE increased the level of LC3-II protein, a marker of autophagy. Knockdown of ATG5 using siRNA inhibited ZFE-induced cytoplasmic vacuolization and cell death. Moreover, in cancer cells that could be induced to undergo cell death by ZFE, the extract increased the phosphorylation of c-Jun N-terminal kinase (JNK), and the JNK inhibitor SP600125 attenuated both vacuolization and cell death. Based on morphology and expression of marker proteins, ZFE-induced cell death was neither apoptosis nor necrosis. Normal intestinal cells were not affected by ZFE. Taken together, our findings show that ZFE induces JNK-dependent ACD, which appears to be the main mechanism underlying its anticancer activity, suggesting a promising starting point for anticancer drug development.

Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

PubMed

Keller, F; Wiemken, A

1982-12-01

The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

Plasmodium falciparum: food vacuole localization of nitric oxide-derived species in intraerythrocytic stages of the malaria parasite

PubMed Central

Ostera, Graciela; Tokumasu, Fuyuki; Oliveira, Fabiano; Sa, Juliana; Furuya, Tetsuya; Teixeira, Clarissa; Dvorak, James

2008-01-01

Nitric oxide (NO) has diverse biological functions. Numerous studies have documented NO’s biosynthetic pathway in a wide variety of organisms. Little is known, however, about NO production in intraerythrocytic Plasmodium falciparum. Using diaminorhodamine-4-methyl acetoxymethylester (DAR-4M AM), a fluorescent indicator, we obtained direct evidence of NO and NO-derived reactive nitrogen species (RNS) production in intraerythrocytic P. falciparum parasites, as well as in isolated food vacuoles from trophozoite stage parasites. We preliminarily identified two gene sequences that might be implicated in NO synthesis in intraerythrocytic P. falciparum. We showed localization of the protein product of one of these two genes, a molecule that is structurally similar to a plant nitrate reductase, in trophozoite food vacuole membranes. We confirmed previous reports on the antiproliferative effect of NOS (nitric oxide synthase) inhibitors in P.falciparum cultures; however, we did not obtain evidence that NOS inhibitors had the ability to inhibit RNS production or that there is an active NOS in mature forms of the parasite. We concluded that a nitrate reductase activity produce NO and NO-derived RNS in or around the food vacuole in P. falciparum parasites. The food vacuole is a critical parasitic compartment involved in hemoglobin degradation, heme detoxification and a target for antimalarial drug action. Characterization of this relatively unexplored synthetic activity could provide important clues into poorly understood metabolic processes of the malaria parasite, PMID:18504040

Reconstructing human pancreatic differentiation by mapping specific cell populations during development

PubMed Central

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-01-01

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2+ population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3, a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated. DOI: http://dx.doi.org/10.7554/eLife.27564.001 PMID:28731406

Death of mitochondria during programmed cell death of leaf mesophyll cells.

PubMed

Selga, Tūrs; Selga, Maija; Pāvila, Vineta

2005-12-01

The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.

Atg5 is Essential for Cellular Immunity in vivo and recruitment of a p47 GTPase to the Toxoplasma gondii Parasitophorous Vacuole in Macrophages

PubMed Central

Zhao, Zijiang; Fux, Blima; Goodwin, Megan; Dunay, Ildiko R.; Strong, David; Miller, Brian C.; Cadwell, Ken; Delgado-Vargas, Monica; Ponpuak, Marisa; Green, Karen G.; Schmidt, Robert E.; Mizushima, Noboru; Deretic, Vojo; Sibley, L. David; Virgin, Herbert W.

2008-01-01

SUMMARY The physiologic importance of autophagy proteins for control of mammalian bacterial and parasitic infection in vivo is unknown. We show that expression of the essential autophagy protein Atg5 in granulocytes and macrophages is required for in vivo resistance to infection with L. monocytogenes and T. gondii. In primary macrophages, Atg5 was not required for IFNγ/LPS-mediated transcription, induction of nitric oxide, or inhibition of T. gondii replication. However, Atg5 was required for IFNγ/LPS-induced damage to the T. gondii parasitophorous vacuole membrane and parasite clearance. While we did not detect autophagosomes enveloping T. gondii, Atg5 was required for recruitment of the IFNγ-inducible p47 GTPase IIGP1 (Irga6) to the vacuole membrane. This work shows that Atg5 expression in phagocytic cells is essential for cellular immunity to intracellular pathogens in vivo and that an autophagy protein can participate in immunity and intracellular killing of pathogens via autophagosome-independent processes such as GTPase trafficking. PMID:18996346

Transforming growth factor (TGF)beta, fibroblast growth factor (FGF) and retinoid signalling pathways promote pancreatic exocrine gene expression in mouse embryonic stem cells.

PubMed Central

Skoudy, Anouchka; Rovira, Meritxell; Savatier, Pierre; Martin, Franz; León-Quinto, Trinidad; Soria, Bernat; Real, Francisco X

2004-01-01

Extracellular signalling cues play a major role in the activation of differentiation programmes. Mouse embryonic stem (ES) cells are pluripotent and can differentiate into a wide variety of specialized cells. Recently, protocols designed to induce endocrine pancreatic differentiation in vitro have been designed but little information is currently available concerning the potential of ES cells to differentiate into acinar pancreatic cells. By using conditioned media of cultured foetal pancreatic rudiments, we demonstrate that ES cells can respond in vitro to signalling pathways involved in exocrine development and differentiation. In particular, modulation of the hedgehog, transforming growth factor beta, retinoid, and fibroblast growth factor pathways in ES cell-derived embryoid bodies (EB) resulted in increased levels of transcripts encoding pancreatic transcription factors and cytodifferentiation markers, as demonstrated by RT-PCR. In EB undergoing spontaneous differentiation, expression of the majority of the acinar genes (i.e. amylase, carboxypeptidase A and elastase) was induced after the expression of endocrine genes, as occurs in vivo during development. These data indicate that ES cells can undergo exocrine pancreatic differentiation with a kinetic pattern of expression reminiscent of pancreas development in vivo and that ES cells can be coaxed to express an acinar phenotype by activation of signalling pathways known to play a role in pancreatic development and differentiation. PMID:14733613

Vacuolating cytotoxin A (VacA), a key toxin for Helicobacter pylori pathogenesis

PubMed Central

Palframan, Samuel L.; Kwok, Terry; Gabriel, Kipros

2012-01-01

More than 50% of the world's population is infected with Helicobacter pylori (H. pylori). Chronic infection with this Gram-negative pathogen is associated with the development of peptic ulcers and is linked to an increased risk of gastric cancer. H. pylori secretes many proteinaceous factors that are important for initial colonization and subsequent persistence in the host stomach. One of the major protein toxins secreted by H. pylori is the Vacuolating cytotoxin A (VacA). After secretion from the bacteria via a type V autotransport secretion system, the 88 kDa VacA toxin (comprised of the p33 and p55 subunits) binds to host cells and is internalized, causing severe “vacuolation” characterized by the accumulation of large vesicles that possess hallmarks of both late endosomes and early lysosomes. The development of “vacuoles” has been attributed to the formation of VacA anion-selective channels in membranes. Apart from its vacuolating effects, it has recently become clear that VacA also directly affects mitochondrial function. Earlier studies suggested that the p33 subunit, but not the p55 subunit of VacA, could enter mitochondria to modulate organelle function. This raised the possibility that a mechanism separate from pore formation may be responsible for the effects of VacA on mitochondria, as crystallography studies and structural modeling predict that both subunits are required for a physiologically stable pore. It has also been suggested that the mitochondrial effects observed are due to indirect effects on pro-apoptotic proteins and direct effects on mitochondrial morphology-related processes. Other studies have shown that both the p55 and p33 subunits can indeed be efficiently imported into mammalian-derived mitochondria raising the possibility that they could re-assemble to form a pore. Our review summarizes and consolidates the recent advances in VacA toxin research, with focus on the outstanding controversies in the field and the key remaining

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift

PubMed Central

Armstrong, Jane A.; Cash, Nicole J.; Ouyang, Yulin; Morton, Jack C.; Chvanov, Michael; Latawiec, Diane; Awais, Muhammad; Tepikin, Alexei V.; Sutton, Robert; Criddle, David N.

2018-01-01

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1–10 μm), whereas higher levels (0.5–1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 μm to 1 mm H2O2), with maximal effects at 500 μm H2O2. H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 μm H2O2. However, higher H2O2 levels (≥50 μm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation. PMID:29626097

Stem Cell-Soluble Signals Enhance Multilumen Formation in SMG Cell Clusters.

PubMed

Maruyama, C L M; Leigh, N J; Nelson, J W; McCall, A D; Mellas, R E; Lei, P; Andreadis, S T; Baker, O J

2015-11-01

Saliva plays a major role in maintaining oral health. Patients with salivary hypofunction exhibit difficulty in chewing and swallowing foods, tooth decay, periodontal disease, and microbial infections. At this time, treatments for hyposalivation are limited to medications (e.g., muscarinic receptor agonists: pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells as well as artificial salivary substitutes. Therefore, advancement of restorative treatments is necessary to improve the quality of life in these patients. Our previous studies indicated that salivary cells are able to form polarized 3-dimensional structures when grown on growth factor-reduced Matrigel. This basement membrane is rich in laminin-III (L1), which plays a critical role in salivary gland formation. Mitotically inactive feeder layers have been used previously to support the growth of many different cell types, as they provide factors necessary for cell growth and organization. The goal of this study was to improve salivary gland cell differentiation in primary cultures by using a combination of L1 and a feeder layer of human hair follicle-derived mesenchymal stem cells (hHF-MSCs). Our results indicated that the direct contact of mouse submandibular (mSMG) cell clusters and hHF-MSCs was not required for mSMG cells to form acinar and ductal structures. However, the hHF-MSC conditioned medium enhanced cell organization and multilumen formation, indicating that soluble signals secreted by hHF-MSCs play a role in promoting these features. © International & American Associations for Dental Research 2015.

Effect of cell density on adipogenic differentiation of mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Hongxu; Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044; Guo, Likun

2009-04-10

The effect of cell density on the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using a patterning technique to induce the formation of a cell density gradient on a micropatterned surface. The adipogenic differentiation of MSCs at a density gradient from 5 x 10{sup 3} to 3 x 10{sup 4} cells/cm{sup 2} was examined. Lipid vacuoles were observed at all cell densities after 1-3 weeks of culture in adipogenic differentiation medium although the lipid vacuoles were scarce at the low cell density and abundant at the high cell density. Real-time RT-PCR analysis showed thatmore » adipogenesis marker genes encoding peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4) were detected in the MSCs cultured at all cell densities. The results suggest that there was no apparent effect of cell density on the adipogenic differentiation of human MSCs.« less

Active penetration of Trypanosoma cruzi into host cells: historical considerations and current concepts

PubMed Central

de Souza, Wanderley; de Carvalho, Tecia M. Ulisses

2013-01-01

In the present short review, we analyze past experiments that addressed the interactions of intracellular pathogenic protozoa (Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium) with host cells and the initial use of the term active penetration to indicate that a protozoan “crossed the host cell membrane, penetrating into the cytoplasm.” However, the subsequent use of transmission electron microscopy showed that, for all of the protozoans and cell types examined, endocytosis, classically defined as involving the formation of a membrane-bound vacuole, took place during the interaction process. As a consequence, the recently penetrated parasites are always within a vacuole, designated the parasitophorous vacuole (PV). PMID:23355838

Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.

PubMed

Chuang, Kun-Han; Liang, Fengshan; Higgins, Ryan; Wang, Yanchang

2016-07-01

Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins. © 2016 Chuang et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae

DOE PAGES

Becker, Argentina; Kannan, T. R.; Taylor, Alexander B.; ...

2015-04-06

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and “walking” pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. In this paper, we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form anmore » overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. Finally, the results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.« less

Deleting the DAG Kinase Dgk1 Augments Yeast Vacuole Fusion Through In-creased Ypt7 Activity and Altered Membrane Fluidity

PubMed Central

Miner, Gregory E.; Starr, Matthew L.; Hurst, Logan R.; Fratti, Rutilio A.

2017-01-01

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P2, and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1. Previously we found that pah1Δ vacuoles were fragmented, blocked in SNARE priming and showed arrested endosomal maturation. In other pathways the effects of deleting PAH1 can be compensated for by additionally deleting DGK1, however deleting both genes did not rescue the pah1Δ vacuolar defects. Deleting DGK1 alone caused a marked increase in vacuole fusion that was attributed to elevated DAG levels. This was accompanied by a gain in resistance to the inhibitory effects of PA as well as inhibitors of Ypt7 activity. Together these data show that Dgk1 function can act as a negative regulator of vacuole fusion through the production of PA at the cost of depleting DAG and reducing Ypt7 activity. PMID:28276191

Mechanisms Involved in Injury and Repair of the Murine Lacrimal Gland: Role of Programmed Cell Death and Mesenchymal Stem Cells

PubMed Central

Zoukhri, Driss

2011-01-01

The non-keratinized epithelia of the ocular surface are constantly challenged by environmental insults, such as smoke, dust, and airborne pathogens. Tears are the sole physical protective barrier for the ocular surface. Production of tears in inadequate quantity or of inadequate quality results in constant irritation of the ocular surface, leading to dry eye disease, also referred to as keratoconjunctivitis sicca (KCS). Inflammation of the lacrimal gland, such as occurs in Sjögren’s syndrome, sarcoidosis, chronic graft versus-host disease, and other pathological conditions, results in inadequate secretion of the aqueous layer of the tear film, and is a leading cause of dry eye disease. The hallmarks of lacrimal gland inflammation are the presence of immune cell infiltrates, loss of acinar epithelial cells (the secreting cells), and increased production of proinflammatory cytokines. To date, the mechanisms leading to acinar cell loss and the associated decline in lacrimal gland secretion are still poorly understood. It is also not understood why the remaining lacrimal gland cells are unable to proliferate in order to regenerate a functioning lacrimal gland. This article reviews recent advances in exocrine tissue injury and repair, with emphasis on the roles of programmed cell death and stem/progenitor cells. PMID:20427009

Electron Microscopy of the Infection and Subsequent Development of Soybean Nodule Cells

PubMed Central

Goodchild, D. J.; Bergersen, F. J.

1966-01-01

Goodchild, D. J. (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia), and F. J. Bergersen. Electron microscopy of the infection and subsequent development of soybean nodule cells. J. Bacteriol. 92:204–213. 1966—Electron microscopy of thin sections of the developing central tissue cells of young soybean root nodules has shown that infection is initiated by a few infection threads which penetrate cells of the young central tissue. Extension growth of the threads may be a result of pressure developed from the growth of the bacteria within the threads. Release of bacteria from a thread is preceded by the development on an infection thread of a bulge with a cellulose-free membrane-bounded extension; bacteria move from this into the host cells by an endocytotic process and remain enclosed in an infection vacuole which is bounded by a membrane of host-cell origin. Multiplication of the intracellular bacteria takes place within these vacuoles. Until the host cell becomes filled with bacteria, the vacuoles separate into discrete units at each division. Later, division of the bacteria occurs within each vacuole, thus leading to the mature structure of the central tissue cells in which several bacteria are enclosed within each membrane-bounded unit. Images PMID:5949564

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole.

PubMed

Tucker, J E; Mauzerall, D; Tucker, E B

1989-07-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

PubMed Central

Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

2015-01-01

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

PubMed

Tourlakis, Marina E; Zhang, Siyi; Ball, Heather L; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S; Guidos, Cynthia J; Durie, Peter R; Rommens, Johanna M

2015-06-01

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b) and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

Effects of Iron Deficiency on Iron Binding and Internalization into Acidic Vacuoles in Dunaliella salina1[W][OA

PubMed Central

Paz, Yakov; Shimoni, Eyal; Weiss, Meira; Pick, Uri

2007-01-01

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe3+ ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability. PMID:17513481

Specific binding of Ulex europaeus agglutinin I lectin to sarcolemma of distal myopathy with rimmed vacuole formation.

PubMed

Yatabe, K; Kawai, M

1997-08-01

Ulex europaeus agglutinin I (UEA I) binding was studied in 83 patients with various neuromuscular disorders. UEA I labelled endomysial capillaries and endothelial cells of perimysial blood vessels in all the examined muscles. There was no UEA I binding to muscle fibres except for all (9) cases of distal myopathy with rimmed vacuole formation (DMRV), 1 of 5 cases of inclusion body myositis and 1 of 36 cases of inflammatory myopathies. The UEA I binding was completely eliminated by preincubation of UEA I solution with L-fucose. Using electron microscopy, the UEA I binding was localized to sarcolemma and intrasarco-plasmic membranous organelles other than mitochondria. Myosatellite cells were not labelled. These findings revealed the existence of fucosylated proteins or lipids in a subset of skeletal muscles suffering from DMRV. Biochemical identification of the fucosylated substance and further detailed study on subcellular localization of UEA I binding may yield important clues to the unknown pathogenesis of DMRV.

Ultrastructural characterisation of the olfactory mucosa of the armadillo Dasypus hybridus (Dasypodidae, Xenarthra)

PubMed Central

FERRARI, C. C.; CARMANCHAHI, P. D.; ALDANA MARCOS, H. J.; AFFANNI, J. M.

2000-01-01

The ultrastructure of the olfactory mucosa of the armadillo Dasypus hybridus was studied. A comparison with the olfactory mucosa of another armadillo (Chaetophractus villosus) was made. The olfactory mucosa of D. hybridus shows many features which are similar to those of other mammals. Interestingly, it differs from the olfactory mucosa of the armadillo C. villosus. A suggestion is made that these differences may be due to differences in the digging habits of these species. In Dasypus, the supporting cells (SCs) showed dense vacuoles, multivesicular bodies and lysosome-like bodies probably related with the endocytotic system. The SCs show a dense network of SER presumably associated with xenobiotic mechanisms. The olfactory receptor neurons exhibit lysosome-like bodies and multivesicular bodies in their perikarya. These organelles suggest the presence of an endocytotic system. Duct cells of Bowman's glands exhibit secretory activities. Bowman's glands are compound-branched tubulo-acinar mixed glands with merocrine secretory mechanisms. PMID:10739023

THE USE OF FORMALDEHYDE-TREATED 131I-ALBUMIN IN THE STUDY OF DIGESTIVE VACUOLES AND SOME PROPERTIES OF THESE PARTICLES FROM MOUSE LIVER

PubMed Central

Mego, John L.; Bertini, Francisco; McQueen, J. Donald

1967-01-01

The trichloroacetic acid-soluble radioactivity released during incubation of mouse liver particles containing intravenously injected formaldehyde-treated 131I-albumin consisted almost entirely of 131I-iodotyrosine. The material was shown to be excreted into the medium and was not due to disruption of the particles by acid. Triton X-100 or the absence of sucrose in the medium inhibited hydrolysis of the particle-associated labeled protein. This inhibition was due to disruption of the digestive vacuoles and dilution of the protein and cathepsins in the suspending medium. These results and other experimental evidence strongly suggest that the 131I-albumin-containing liver particles are digestive vacuoles. The results also establish that 131I-albumin may be used to study these vacuoles. High concentrations of sucrose (1 M) inhibited degradation of intraparticulate protein. However, 1 M salts inhibited only the rate of the digestion. Sucrose had an inhibitory effect on a crude cathepsin preparation, and salts stimulated the activity when 131I-albumin was used as substrate. The effect of high sucrose concentrations as an inhibitor of protein hydrolysis within digestive vacuoles was, therefore, most likely due principally to an inhibition of cathepsin activity within the vacuoles. The effect of salt was probably caused by a stimulation of both intra- and extra-particulate cathepsin activities, although 0.5–1.0 M KCl appeared to protect the particles. PMID:6034485

Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations.

PubMed

Viktorinová, Ivana; Henry, Ian; Tomancak, Pavel

2017-11-01

Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs.

Epithelial rotation is preceded by planar symmetry breaking of actomyosin and protects epithelial tissue from cell deformations

PubMed Central

Henry, Ian; Tomancak, Pavel

2017-01-01

Symmetry breaking is involved in many developmental processes that form bodies and organs. One of them is the epithelial rotation of developing tubular and acinar organs. However, how epithelial cells move, how they break symmetry to define their common direction, and what function rotational epithelial motions have remains elusive. Here, we identify a dynamic actomyosin network that breaks symmetry at the basal surface of the Drosophila follicle epithelium of acinar-like primitive organs, called egg chambers, and may represent a candidate force-generation mechanism that underlies the unidirectional motion of this epithelial tissue. We provide evidence that the atypical cadherin Fat2, a key planar cell polarity regulator in Drosophila oogenesis, directs and orchestrates transmission of the intracellular actomyosin asymmetry cue onto a tissue plane in order to break planar actomyosin symmetry, facilitate epithelial rotation in the opposite direction, and direct the elongation of follicle cells. In contrast, loss of this rotational motion results in anisotropic non-muscle Myosin II pulses that are disorganized in plane and causes cell deformations in the epithelial tissue of Drosophila eggs. Our work demonstrates that atypical cadherins play an important role in the control of symmetry breaking of cellular mechanics in order to facilitate tissue motion and model epithelial tissue. We propose that their functions may be evolutionarily conserved in tubular/acinar vertebrate organs. PMID:29176774

Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

PubMed Central

Winter, Gal; Cordente, Antonio G.; Curtin, Chris

2014-01-01

Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes. PMID:25517415

Osimertinib induces autophagy and apoptosis via reactive oxygen species generation in non-small cell lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Zheng-Hai; Cao, Wen-Xiang; Su, Min-Xia

Osimertinib (OSI), also known as AZD9291, is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has been approved for the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR T790M mutation. Herein, we indicated for the first time that OSI increased the accumulations of cytoplasmic vacuoles, the expression of phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II), and the formation of GFP-LC3 puncta in various cancer cells. The OSI-induced expression of LC3-II was further increased when combined treatment with chloroquine (CQ), an autophagy inhibitor, and the mRFP-EGFP-LC3 plasmid-transfected cells exposed to OSI led to the production of moremore » red-fluorescent puncta than green-fluorescent puncta, indicating OSI induced autophagic flux in the NSCLC cells. Knockdown of EGFR showed no effect on the OSI-induced expression of LC3-II in NCI-H1975 cells. In addition, OSI increased reactive oxygen species (ROS) generation and scavenge of ROS via pretreatment with N-acetyl-L-cysteine (NAC), catalase (CAT), or vitamin E (Vita E) significantly inhibited OSI-induced the accumulations of cytoplasmic vacuoles, the expression of LC3-II, as well as the formation of GFP-LC3 puncta. Combinative treatment with CQ could not remarkably change the OSI-induced cell viability decrease, whereas the OSI-induced cell viability decrease and apoptosis could be reversed through pretreatment with NAC, CAT, and Vita E, respectively. Taken together, this is the first report that OSI induces an accompanied autophagy and the generation of ROS is critical for the OSI-induced autophagy, cell viability decrease, and apoptosis in NSCLC cells. - Highlights: • Osimertinib induced the expressions of cytoplasmic vacuoles and autophagic markers in different cancer cells. • Osimertinib induced autophagic flux in NSCLC NCI-H1975 and HCC827 cell lines. • ROS generation contributed to osimertinib

The shoot meristem identity gene TFL1 is involved in flower development and trafficking to the protein storage vacuole

PubMed Central

Sohn, Eun Ju; Rojas-Pierce, Marcela; Pan, Songqin; Carter, Clay; Serrano-Mislata, Antonio; Madueño, Francisco; Rojo, Enrique; Surpin, Marci; Raikhel, Natasha V.

2007-01-01

Plants are unique in their ability to store proteins in specialized protein storage vacuoles (PSVs) within seeds and vegetative tissues. Although plants use PSV proteins during germination, before photosynthesis is fully functional, the roles of PSVs in adult vegetative tissues are not understood. Trafficking pathways to PSVs and lytic vacuoles appear to be distinct. Lytic vacuoles are analogous evolutionarily to yeast and mammalian lysosomes. However, it is unclear whether trafficking to PSVs has any analogy to pathways in yeast or mammals, nor is PSV ultrastructure known in Arabidopsis vegetative tissue. Therefore, alternative approaches are required to identify components of this pathway. Here, we show that an Arabidopsis thaliana mutant that disrupts PSV trafficking identified TERMINAL FLOWER 1 (TFL1), a shoot meristem identity gene. The tfl1-19/mtv5 (for “modified traffic to the vacuole”) mutant is specifically defective in trafficking of proteins to the PSV. TFL1 localizes to endomembrane compartments and colocalizes with the putative δ-subunit of the AP-3 adapter complex. Our results suggest a developmental role for the PSV in vegetative tissues. PMID:18003908

Regeneration and Repair of the Exocrine Pancreas

PubMed Central

Murtaugh, L. Charles; Keefe, Matthew

2015-01-01

Pancreatitis is caused by inflammatory injury to the exocrine pancreas, from which both humans and animal models appear to recover via regeneration of digestive enzyme-producing acinar cells. This regenerative process involves transient phases of inflammation, metaplasia and redifferentiation, driven by cell-cell interactions between acinar cells, leukocytes and resident fibroblasts. The NFκB signaling pathway is a critical determinant of pancreatic inflammation and metaplasia, whereas a number of developmental signals and transcription factors are devoted to promoting acinar redifferentiation after injury. Imbalances between these pro-inflammatory and pro-differentiation pathways contribute to chronic pancreatitis, characterized by persistent inflammation, fibrosis and acinar dedifferentiation. Loss of acinar cell differentiation also drives pancreatic cancer initiation, providing a mechanistic link between pancreatitis and cancer risk. Unraveling the molecular bases of exocrine regeneration may identify new therapeutic targets for treatment and prevention of both of these deadly diseases. PMID:25386992

The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components

PubMed Central

Fischer, Martina; Jehmlich, Nico; Rose, Laura; Koch, Sophia; Laue, Michael; Renard, Bernhard Y.; Schmidt, Frank; Heuer, Dagmar

2015-01-01

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells’ antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection. PMID:26042774

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy[OPEN

PubMed Central

2017-01-01

Turnover of dysfunctional organelles is vital to maintain homeostasis in eukaryotic cells. As photosynthetic organelles, plant chloroplasts can suffer sunlight-induced damage. However, the process for turnover of entire damaged chloroplasts remains unclear. Here, we demonstrate that autophagy is responsible for the elimination of sunlight-damaged, collapsed chloroplasts in Arabidopsis thaliana. We found that vacuolar transport of entire chloroplasts, termed chlorophagy, was induced by UV-B damage to the chloroplast apparatus. This transport did not occur in autophagy-defective atg mutants, which exhibited UV-B-sensitive phenotypes and accumulated collapsed chloroplasts. Use of a fluorescent protein marker of the autophagosomal membrane allowed us to image autophagosome-mediated transport of entire chloroplasts to the central vacuole. In contrast to sugar starvation, which preferentially induced distinct type of chloroplast-targeted autophagy that transports a part of stroma via the Rubisco-containing body (RCB) pathway, photooxidative damage induced chlorophagy without prior activation of RCB production. We further showed that chlorophagy is induced by chloroplast damage caused by either artificial visible light or natural sunlight. Thus, this report establishes that an autophagic process eliminates entire chloroplasts in response to light-induced damage. PMID:28123106

Pancreatic β-cell regeneration: Facultative or dedicated progenitors?

PubMed

Afelik, Solomon; Rovira, Meritxell

2017-04-15

The adult pancreas is only capable of limited regeneration. Unlike highly regenerative tissues such as the skin, intestinal crypts and hematopoietic system, no dedicated adult stem cells or stem cell niche have so far been identified within the adult pancreas. New β cells have been shown to form in the adult pancreas, in response to high physiological demand or experimental β-cell ablation, mostly by replication of existing β cells. The possibility that new β cells are formed from other sources is currently a point of major controversy. Under particular injury conditions, fully differentiated pancreatic duct and acinar cells have been shown to dedifferentiate into a progenitor-like state, however the extent, to which ductal, acinar or other endocrine cells contribute to restoring pancreatic β-cell mass remains to be resolved. In this review we focus on regenerative events in the pancreas with emphasis on the restoration of β-cell mass. We present an overview of regenerative responses noted within the different pancreatic lineages, following injury. We also highlight the intrinsic plasticity of the adult pancreas that allows for inter-conversion of fully differentiated pancreatic lineages through manipulation of few genes or growth factors. Taken together, evidence from a number of studies suggest that differentiated pancreatic lineages could act as facultative progenitor cells, but the extent to which these contribute to β-cell regeneration in vivo is still a matter of contention. Copyright © 2016. Published by Elsevier B.V.

Multinodular and vacuolating neuronal tumors of the cerebrum: 10 cases of a distinctive seizure-associated lesion.

PubMed

Huse, Jason T; Edgar, Mark; Halliday, John; Mikolaenko, Irina; Lavi, Ehud; Rosenblum, Marc K

2013-09-01

We report 10 cases of a non-neurocytic, purely neuronal tumor affecting adults. Situated in the cerebral hemispheres, with 7 of 10 confined to the temporal lobes, most presented with seizures as their principal clinical manifestations. On magnetic resosnance imaging (MRI), the tumors generally appeared solid and non-contrast enhancing with minimal diffuse infiltration, edema, or mass effect. Six examples demonstrated internal nodularity. Microscopically, the tumor cells were largely distributed into discrete and coalescent nodules exhibiting varying degrees of matrix vacuolization, principally within the deep cortical ribbon and superficial subcortical white matter. Populating elements ranged from morphologically ambiguous to recognizably neuronal, with only two cases manifesting overt ganglion cell cytology. In all cases, tumor cells exhibited widespread nuclear immunolabeling for the HuC/HuD neuronal antigens, although expression of other neuronal markers, including synaptophysin, neurofilament and chromogranin was variable to absent. Tumor cells also failed to express GFAP, p53, IDH1 R132H, or CD34, although CD34-labeling ramified neural elements were present in the adjoining cortex of seven cases. Molecular analysis in a subset of cases failed to reveal DNA copy number abnormalities or BRAF V600E mutation. Follow-up data indicate that this unusual neuronal lesion behaves in benign, World Health Organization (WHO) grade I fashion and is amenable to surgical control. © 2013 International Society of Neuropathology.

Histopathology and pathogenesis of caerulein-, duct ligation-, and arginine-induced acute pancreatitis in Sprague-Dawley rats and C57BL6 mice.

PubMed

Zhang, Jun; Rouse, Rodney L

2014-09-01

Three classical rodent models of acute pancreatitis were created in an effort to identify potential pre-clinical models of drug-induced pancreatitis (DIP) and candidate non-invasive biomarkers for improved detection of DIP. Study objectives included designing a lexicon to minimize bias by capturing normal variation and spontaneous and injury-induced changes while maintaining the ability to statistically differentiate degrees of change, defining morphologic anchors for novel pancreatic injury biomarkers, and improved understanding of mechanisms responsible for pancreatitis. Models were created in male Sprague-Dawley rats and C57BL6 mice through: 1) administration of the cholecystokinin analog, caerulein; 2) administration of arginine; 3) surgical ligation of the pancreatic duct. Nine morphologically detectable processes were used in the lexicon; acinar cell hypertrophy; acinar cell autophagy; acinar cell apoptosis; acinar cell necrosis; vascular injury; interstitial edema, inflammation and hemorrhage; fat necrosis; ductal changes; acinar cell atrophy. Criteria were defined for scoring levels (0 = absent, 1 = mild, 2 = moderate, 3 = severe) for each lexicon component. Consistent with previous studies, histopathology scores were significant greater in rats compared to mice at baseline and after treatment. The histopathology scores in caerulein and ligation-treated rats and mice were significantly greater than those of arginine-treated rats and mice. The present study supports a multifaceted pathogenesis for acute pancreatitis in which intra-acinar trypsinogen activation, damage to acinar cells, fat cells, and vascular cells as well as activation/degranulation of mast cells and activated macrophages all contribute to the initiation and/or progression of acute inflammation of the exocrine pancreas.

Transport of Stachyose and Sucrose by Vacuoles of Japanese Artichoke (Stachys sieboldii) Tubers 1

PubMed Central

Keller, Felix

1992-01-01

Vacuoles are the stores for large amounts of stachyose [αgal (1,6) αgal (1,6) αglc (1,2) βfru] in tubers of Japanese artichoke (Stachys sieboldii). The uptake of stachyose by these vacuoles was examined and compared with that of sucrose. The uptake mechanisms of both sugars were quite similar. The kinetics showed a single saturable response to increasing external concentrations of 14C-sugars with similar apparent Km values of about 50 and 30 millimolar for stachyose and sucrose, respectively. The uptake rates, however, were always higher for stachyose than for sucrose. Stachyose and sucrose uptake was inhibited by fructose and raffinose, and, reciprocally, by sucrose and stachyose, but not by glucose or galactose. The main structural feature common to all sugars recognized by the uptake systems seems to be a terminal fructosyl residue. The uptake of both sugars was stimulated by Mg-ATP and inorganic pyrophosphate, suggesting a proton-sugar antiport system. The possibility that stachyose and sucrose might be transported by the same carrier is discussed. PMID:16668659

Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

PubMed

Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael

2014-11-01

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease. Copyright © 2014 Elsevier GmbH. All rights reserved.

The vacuole system is a significant intracellular pathway for longitudinal solute transport in basidiomycete fungi.

PubMed

Darrah, P R; Tlalka, M; Ashford, A; Watkinson, S C; Fricker, M D

2006-07-01

Mycelial fungi have a growth form which is unique among multicellular organisms. The data presented here suggest that they have developed a unique solution to internal solute translocation involving a complex, extended vacuole. In all filamentous fungi examined, this extended vacuole forms an interconnected network, dynamically linked by tubules, which has been hypothesized to act as an internal distribution system. We have tested this hypothesis directly by quantifying solute movement within the organelle by photobleaching a fluorescent vacuolar marker. Predictive simulation models were then used to determine the transport characteristics over extended length scales. This modeling showed that the vacuolar organelle forms a functionally important, bidirectional diffusive transport pathway over distances of millimeters to centimeters. Flux through the pathway is regulated by the dynamic tubular connections involving homotypic fusion and fission. There is also a strongly predicted interaction among vacuolar organization, predicted diffusion transport distances, and the architecture of the branching colony margin.

Immunohistochemical localization of Clara cell secretory proteins (CC10-CC26) and Annexin-1 protein in rat major salivary glands

PubMed Central

Cecchini, Maria Paola; Merigo, Flavia; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

2009-01-01

The oral cavity is continuously bathed by saliva secreted by the major and minor salivary glands. Saliva is the first biological medium to confront external materials that are taken into the body as part of food or drink or inhaled volatile substances, and it contributes to the first line of oral defence. In humans, it has been shown that sputum and a variety of biological fluids contain Clara cell secretory proteins (CC10–CC26). Various studies of the respiratory apparatus have suggested their protective effect against inflammatory response and oxidative stress. Recently, CC10 deficiency has been related to the protein Annexin-1 (ANXA1), which has immunomodulatory and anti-inflammatory properties. Considering the defensive role of both Clara cell secretory proteins and ANXA1 in the respiratory apparatus, and the importance of salivary gland secretion in the first line of oral defence, we decided to evaluate the expression of CC10, CC26 and ANXA1 proteins in rat major salivary glands using immunohistochemistry. CC10 expression was found only in the ductal component of the sublingual gland. Parotid and submandibular glands consistently lacked CC10 immunoreactivity. In the parotid gland, both acinar and ductal cells were always CC26-negative, whereas in the submandibular gland, immunostaining was localized in the ductal component and in the periodic acid Schiff (PAS)-positive area. In the sublingual gland, ductal cells were always positive. Acinar cells were not immunostained at all. ANXA1 was expressed in ductal cells in all three major glands. In parotid and sublingual glands, acinar cells were negative. In submandibular glands, immunostaining was present in the mucous PAS-positive portion, whereas serous acinar cells were consistently negative. The existence of some CC10-CC26–ANXA1-positive cells in rat salivary glandular tissue is an interesting preliminary finding which could support the hypothesis, suggested for airway tissue, that these proteins have a

Vacuolar processing enzyme: an executor of plant cell death.

PubMed

Hara-Nishimura, Ikuko; Hatsugai, Noriyuki; Nakaune, Satoru; Kuroyanagi, Miwa; Nishimura, Mikio

2005-08-01

Apoptotic cell death in animals is regulated by cysteine proteinases called caspases. Recently, vacuolar processing enzyme (VPE) was identified as a plant caspase. VPE deficiency prevents cell death during hypersensitive response and cell death of limited cell layers at the early stage of embryogenesis. Because plants do not have macrophages, dying cells must degrade their materials by themselves. VPE plays an essential role in the regulation of the lytic system of plants during the processes of defense and development. VPE is localized in the vacuoles, unlike animal caspases, which are localized in the cytosol. Thus, plants might have evolved a regulated cellular suicide strategy that, unlike animal apoptosis, is mediated by VPE and the vacuoles.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

PubMed Central

Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

2015-01-01

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole 1

PubMed Central

Tucker, Joseph E.; Mauzerall, David; Tucker, Edward B.

1989-01-01

The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water. PMID:16666864

Hyaluronan synthesis in cultured tobacco cells (BY-2) expressing a chlorovirus enzyme: cytological studies.

PubMed

Rakkhumkaew, Numfon; Shibatani, Shigeo; Kawasaki, Takeru; Fujie, Makoto; Yamada, Takashi

2013-04-01

Extraction of hyaluronan from animals or microbial fermentation has risks including contamination with pathogens and microbial toxins. In this work, tobacco cultured-cells (BY-2) were successfully transformed with a chloroviral hyaluronan synthase (cvHAS) gene to produce hyaluronan. Cytological studies revealed accumulation of HA on the cells, and also in subcellular fractions (protoplasts, miniplasts, vacuoplasts, and vacuoles). Transgenic BY-2 cells harboring a vSPO-cvHAS construct containing the vacuolar targeting signal of sporamin connected to the N-terminus of cvHAS accumulated significant amounts of HA in vacuoles. These results suggested that cvHAS successfully functions on the vacuolar membrane and synthesizes/transports HA into vacuoles. Efficient synthesis of HA using this system provides a new method for practical production of HA. Copyright © 2012 Wiley Periodicals, Inc.

Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

PubMed Central

Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

2016-01-01

Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells. PMID:27916824

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me–induced apoptosis in breast cancer cells

PubMed Central

Lee, A Reum; Yoon, Mi Jin; Cho, Hyeseong; Lee, Jong-Soo; Choi, Kyeong Sook

2015-01-01

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me–induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me–induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me–induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells. PMID:26053096

Ca2+ influx-mediated dilation of the endoplasmic reticulum and c-FLIPL downregulation trigger CDDO-Me-induced apoptosis in breast cancer cells.

PubMed

Jeong, Soo Ah; Kim, In Young; Lee, A Reum; Yoon, Mi Jin; Cho, Hyeseong; Lee, Jong-Soo; Choi, Kyeong Sook

2015-08-28

The synthetic triterpenoid 2-cyano-3, 12-dioxooleana-1, 9(11)-dien-C28-methyl ester (CDDO-Me) is considered a promising anti-tumorigenic compound. In this study, we show that treatment with CDDO-Me induces progressive endoplasmic reticulum (ER)-derived vacuolation in various breast cancer cells and ultimately kills these cells by inducing apoptosis. We found that CDDO-Me-induced increases in intracellular Ca2+ levels, reflecting influx from the extracellular milieu, make a critical contribution to ER-derived vacuolation and subsequent cell death. In parallel with increasing Ca2+ levels, CDDO-Me markedly increased the generation of reactive oxygen species (ROS). Interestingly, there exists a reciprocal positive-regulatory loop between Ca2+ influx and ROS generation that triggers ER stress and ER dilation in response to CDDO-Me. In addition, CDDO-Me rapidly reduced the protein levels of c-FLIPL (cellular FLICE-inhibitory protein) and overexpression of c-FLIPL blocked CDDO-Me-induced cell death, but not vacuolation. These results suggest that c-FLIPL downregulation is a key contributor to CDDO-Me-induced apoptotic cell death, independent of ER-derived vacuolation. Taken together, our results show that ER-derived vacuolation via Ca2+ influx and ROS generation as well as caspase activation via c-FLIPL downregulation are responsible for the potent anticancer effects of CDDO-Me on breast cancer cells.

AMPK and vacuole-associated Atg14p orchestrate μ-lipophagy for energy production and long-term survival under glucose starvation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, Arnold Y.; Lau, Pick -Wei; Feliciano, Daniel

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interactedmore » with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.« less

AMPK and vacuole-associated Atg14p orchestrate μ-lipophagy for energy production and long-term survival under glucose starvation

DOE PAGES

Seo, Arnold Y.; Lau, Pick -Wei; Feliciano, Daniel; ...

2017-04-10

Dietary restriction increases the longevity of many organisms, but the cell signaling and organellar mechanisms underlying this capability are unclear. We demonstrate that to permit long-term survival in response to sudden glucose depletion, yeast cells activate lipid-droplet (LD) consumption through micro-lipophagy (µ-lipophagy), in which fat is metabolized as an alternative energy source. AMP-activated protein kinase (AMPK) activation triggered this pathway, which required Atg14p. More gradual glucose starvation, amino acid deprivation or rapamycin did not trigger µ-lipophagy and failed to provide the needed substitute energy source for long-term survival. During acute glucose restriction, activated AMPK was stabilized from degradation and interactedmore » with Atg14p. This prompted Atg14p redistribution from ER exit sites onto liquid-ordered vacuole membrane domains, initiating µ-lipophagy. Our findings that activated AMPK and Atg14p are required to orchestrate µ-lipophagy for energy production in starved cells is relevant for studies on aging and evolutionary survival strategies of different organisms.« less

Scolopendra subspinipes mutilans protected the cerulein-induced acute pancreatitis by inhibiting high-mobility group box protein-1.

PubMed

Jo, Il-Joo; Bae, Gi-Sang; Park, Kyoung-Chel; Choi, Sun Bok; Jung, Won-Seok; Jung, Su-Young; Cho, Jung-Hee; Choi, Mee-Ok; Song, Ho-Joon; Park, Sung-Joo

2013-03-14

To evaluate the inhibitory effects of Scolopendra subspinipes mutilans (SSM) on cerulein-induced acute pancreatitis (AP) in a mouse model. SSM water extract (0.1, 0.5, or 1 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein. Once AP developed, the stable cholecystokinin analogue, cerulein was injected hourly, over a 6 h period. Blood samples were taken 6 h later to determine serum amylase, lipase, and cytokine levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. To specify the role of SSM in pancreatitis, the pancreatic acinar cells were isolated using collagenase method. Then the cells were pre-treated with SSM, then stimulated with cerulein. The cell viability, cytokine productions and high-mobility group box protein-1 (HMGB-1) were measured. Furthermore, the regulating mechanisms of SSM action were evaluated. The administration of SSM significantly attenuated the severity of pancreatitis and pancreatitis associated lung injury, as was shown by the reduction in pancreatic edema, neutrophil infiltration, vacuolization and necrosis. SSM treatment also reduced pancreatic weight/body weight ratio, serum amylase, lipase and cytokine levels, and mRNA expression of multiple inflammatory mediators such as tumor necrosis factor-α and interleukin-1β. In addition, treatment with SSM inhibited HMGB-1 expression in the pancreas during AP. In accordance with in vivo data, SSM inhibited the cerulein-induced acinar cell death, cytokine, and HMGB-1 release. SSM also inhibited the activation of c-Jun NH2-terminal kinase, p38 and nuclear factor (NF)-κB. These results suggest that SSM plays a protective role during the development of AP and pancreatitis associated lung injury via deactivating c-Jun NH2-terminal kinase, p38 and NF-κB.

Scolopendra subspinipes mutilans protected the cerulein-induced acute pancreatitis by inhibiting high-mobility group box protein-1

PubMed Central

Jo, Il-Joo; Bae, Gi-Sang; Park, Kyoung-Chel; Choi, Sun Bok; Jung, Won-Seok; Jung, Su-Young; Cho, Jung-Hee; Choi, Mee-Ok; Song, Ho-Joon; Park, Sung-Joo

2013-01-01

AIM: To evaluate the inhibitory effects of Scolopendra subspinipes mutilans (SSM) on cerulein-induced acute pancreatitis (AP) in a mouse model. METHODS: SSM water extract (0.1, 0.5, or 1 g/kg) was administrated intraperitoneally 1 h prior to the first injection of cerulein. Once AP developed, the stable cholecystokinin analogue, cerulein was injected hourly, over a 6 h period. Blood samples were taken 6 h later to determine serum amylase, lipase, and cytokine levels. The pancreas and lungs were rapidly removed for morphological examination, myeloperoxidase assay, and real-time reverse transcription polymerase chain reaction. To specify the role of SSM in pancreatitis, the pancreatic acinar cells were isolated using collagenase method. Then the cells were pre-treated with SSM, then stimulated with cerulein. The cell viability, cytokine productions and high-mobility group box protein-1 (HMGB-1) were measured. Furthermore, the regulating mechanisms of SSM action were evaluated. RESULTS: The administration of SSM significantly attenuated the severity of pancreatitis and pancreatitis associated lung injury, as was shown by the reduction in pancreatic edema, neutrophil infiltration, vacuolization and necrosis. SSM treatment also reduced pancreatic weight/body weight ratio, serum amylase, lipase and cytokine levels, and mRNA expression of multiple inflammatory mediators such as tumor necrosis factor-α and interleukin-1β. In addition, treatment with SSM inhibited HMGB-1 expression in the pancreas during AP. In accordance with in vivo data, SSM inhibited the cerulein-induced acinar cell death, cytokine, and HMGB-1 release. SSM also inhibited the activation of c-Jun NH2-terminal kinase, p38 and nuclear factor (NF)-κB. CONCLUSION: These results suggest that SSM plays a protective role during the development of AP and pancreatitis associated lung injury via deactivating c-Jun NH2-terminal kinase, p38 and NF-κB. PMID:23539679

Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting.

PubMed

Suzuki, Atsushi; Nakauchi, Hiromitsu; Taniguchi, Hideki

2004-08-01

During pancreatic development, neogenesis, and regeneration, stem cells might act as a central player to generate endocrine, acinar, and duct cells. Although these cells are well known as pancreatic stem cells (PSCs), indisputable proof of their existence has not been reported. Identification of phenotypic markers for PSCs leads to their prospective isolation and precise characterization to clear whether stem cells exist in the pancreas. By combining flow cytometry and clonal analysis, we show here that a possible pancreatic stem or progenitor cell candidate that resides in the developing and adult mouse pancreas expresses the receptor for the hepatocyte growth factor (HGF) c-Met, but does not express hematopoietic and vascular endothelial antigens such as CD45, TER119, c-Kit, and Flk-1. These cells formed clonal colonies in vitro and differentiated into multiple pancreatic lineage cells from single cells. Some of them could largely expand with self-renewing cell divisions in culture, and, following cell transplantation, they differentiated into pancreatic endocrine and acinar cells in vivo. Furthermore, they produced cells expressing multiple markers of nonpancreatic organs including liver, stomach, and intestine in vitro. Our data strongly suggest that c-Met/HGF signaling plays an important role in stem/progenitor cell function in both developing and adult pancreas. By using this antigen, PSCs could be isolated prospectively, enabling a detailed investigation of stem cell markers and application toward regenerative therapies for diabetes.

Influence of KDEL on the fate of trimeric or assembly-defective phaseolin: selective use of an alternative route to vacuoles.

PubMed

Frigerio, L; Pastres, A; Prada, A; Vitale, A

2001-05-01

The tetrapeptide KDEL is commonly found at the C terminus of soluble proteins of the endoplasmic reticulum (ER), and it contributes to their localization by interacting with a receptor that recycles between the Golgi complex and the ER. We investigated the effects of the addition of KDEL to phaseolin, a protein normally delivered from the ER to storage vacuoles via the Golgi complex. We show that KDEL prevents acquisition of trans-Golgi-specific glycan modifications and causes interactions with the chaperone BiP that are distinct from the ones between BiP and defective proteins. KDEL markedly increases the stability of phaseolin, but a small proportion of phaseolin-KDEL slowly reaches the vacuole without undergoing Golgi-mediated glycan modifications, in a process that can be inhibited by brefeldin A but not monensin. Our results indicate that KDEL can operate with high efficiency before proteins can reach the late Golgi cisternae but allows or promotes delivery to vacuoles via an alternative mechanism. However, addition of KDEL does not alter the destiny of an assembly-defective form of phaseolin, suggesting that the plant ER quality control mechanism is dominant over KDEL effects.

Relationship between carbachol hyperstimulation-induced pancreatic intracellular trypsinogen and NF-kappa B activation in rats in vitro.

PubMed

Jiang, Chunfang; Zheng, Hai; Liu, Sunan; Fang, Kaifeng

2008-02-01

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Loss of notochordal cell phenotype in 3D-cell cultures: implications for disc physiology and disc repair.

PubMed

Omlor, G W; Nerlich, A G; Tirlapur, U K; Urban, J P; Guehring, T

2014-12-01

Embryonic notochordal disc nucleus cells (NC) have been identified to protect disc tissue against disc degeneration but in human beings NC phenotype gets lost with aging and the pathophysiological mechanisms are poorly understood. NC may stimulate other cells via soluble factors, and NC-conditioned medium can be used to stimulate matrix production of other disc cells and mesenchymal stem cells and thus may be of special interest for biological disc repair. As this stimulatory effect is associated with the NC phenotype, we investigated how cell morphology and gene-expression of the NC phenotype changes with time in 3D-cell culture. NC and inner annulus chondrocyte-like cells (CLC) from immature pigtails (freshly isolated cells/tissue, 3D-alginate beads, 3D-clusters) were cultured for up to 16 days under normoxia and hypoxia. Protein-expression was analysed by immunohistology and gene-expression analysis was carried out on freshly isolated cells and cultured cells. Cell morphology and proliferation were analysed by two-photon-laser-microscopy. Two-photon-laser-microscopy showed a homogenous and small CLC population in the inner annulus, which differed from the large vacuole-containing NC in the nucleus. Immunohistology found 93 % KRT8 positive cells in the nucleus and intracellular and pericellular Col2, IL6, and IL12 staining while CLC were KRT8 negative. Freshly isolated NC showed significantly higher KRT8 and CAIII but lower Col2 gene-expression than CLC. NC in 3D-cultures demonstrated significant size reduction and loss of vacuoles with culture time, all indicating a loss of the characteristic NC morphology. Hypoxia reduced the rate of decrease in NC size and vacuoles. Gene-expression of KRT8 and CAIII in NC fell significantly early in culture while Col2 did not decrease significantly within the culture period. In CLC, KRT8 and CAIII gene-expression was low and did not change noticeably in culture, whereas Col2 expression fell with time in culture. 3D

Morphometric Characterization of Rat and Human Alveolar Macrophage Cell Models and their Response to Amiodarone using High Content Image Analysis.

PubMed

Hoffman, Ewelina; Patel, Aateka; Ball, Doug; Klapwijk, Jan; Millar, Val; Kumar, Abhinav; Martin, Abigail; Mahendran, Rhamiya; Dailey, Lea Ann; Forbes, Ben; Hutter, Victoria

2017-12-01

Progress to the clinic may be delayed or prevented when vacuolated or "foamy" alveolar macrophages are observed during non-clinical inhalation toxicology assessment. The first step in developing methods to study this response in vitro is to characterize macrophage cell lines and their response to drug exposures. Human (U937) and rat (NR8383) cell lines and primary rat alveolar macrophages obtained by bronchoalveolar lavage were characterized using high content fluorescence imaging analysis quantification of cell viability, morphometry, and phospholipid and neutral lipid accumulation. Cell health, morphology and lipid content were comparable (p < 0.05) for both cell lines and the primary macrophages in terms of vacuole number, size and lipid content. Responses to amiodarone, a known inducer of phospholipidosis, required analysis of shifts in cell population profiles (the proportion of cells with elevated vacuolation or lipid content) rather than average population data which was insensitive to the changes observed. A high content image analysis assay was developed and used to provide detailed morphological characterization of rat and human alveolar-like macrophages and their response to a phospholipidosis-inducing agent. This provides a basis for development of assays to predict or understand macrophage vacuolation following inhaled drug exposure.

Regulation of Breast Cancer Stem Cell by Tissue Rigidity

DTIC Science & Technology

2014-06-01

pose the similar question as Paszek et al but in a more biomimetic niche: “Does the mature mammary acinar structure desensitize mammary epithelial...2728032. 6. Lucero HA, Kagan HM. Lysyl oxidase: an oxidative enzyme and effector of cell function. Cell Mol Life Sci. 2006;63(19-20):2304-16. 7. Levental...requirement of EMT and/or MET during the individual steps of tumor metastasis and discuss the potential of targeting this program when treating

Niemann-Pick type C proteins promote microautophagy by expanding raft-like membrane domains in the yeast vacuole

PubMed Central

Tsuji, Takuma; Fujimoto, Megumi; Tatematsu, Tsuyako; Cheng, Jinglei; Orii, Minami; Takatori, Sho; Fujimoto, Toyoshi

2017-01-01

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole. DOI: http://dx.doi.org/10.7554/eLife.25960.001 PMID:28590904

Fierce Competition between Toxoplasma and Chlamydia for Host Cell Structures in Dually Infected Cells

PubMed Central

Romano, Julia D.; de Beaumont, Catherine; Carrasco, Jose A.; Ehrenman, Karen; Bavoil, Patrik M.

2013-01-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients. PMID:23243063

Fierce competition between Toxoplasma and Chlamydia for host cell structures in dually infected cells.

PubMed

Romano, Julia D; de Beaumont, Catherine; Carrasco, Jose A; Ehrenman, Karen; Bavoil, Patrik M; Coppens, Isabelle

2013-02-01

The prokaryote Chlamydia trachomatis and the protozoan Toxoplasma gondii, two obligate intracellular pathogens of humans, have evolved a similar modus operandi to colonize their host cell and salvage nutrients from organelles. In order to gain fundamental knowledge on the pathogenicity of these microorganisms, we have established a cell culture model whereby single fibroblasts are coinfected by C. trachomatis and T. gondii. We previously reported that the two pathogens compete for the same nutrient pools in coinfected cells and that Toxoplasma holds a significant competitive advantage over Chlamydia. Here we have expanded our coinfection studies by examining the respective abilities of Chlamydia and Toxoplasma to co-opt the host cytoskeleton and recruit organelles. We demonstrate that the two pathogen-containing vacuoles migrate independently to the host perinuclear region and rearrange the host microtubular network around each vacuole. However, Toxoplasma outcompetes Chlamydia to the host microtubule-organizing center to the detriment of the bacterium, which then shifts to a stress-induced persistent state. Solely in cells preinfected with Chlamydia, the centrosomes become associated with the chlamydial inclusion, while the Toxoplasma parasitophorous vacuole displays growth defects. Both pathogens fragment the host Golgi apparatus and recruit Golgi elements to retrieve sphingolipids. This study demonstrates that the productive infection by both Chlamydia and Toxoplasma depends on the capability of each pathogen to successfully adhere to a finely tuned developmental program that aims to remodel the host cell for the pathogen's benefit. In particular, this investigation emphasizes the essentiality of host organelle interception by intravacuolar pathogens to facilitate access to nutrients.

Isolation and Propagation of Mesenchymal Stem Cells from the Lacrimal Gland

PubMed Central

You, Samantha; Kublin, Claire L.; Avidan, Orna; Miyasaki, David

2011-01-01

Purpose. Previously, it was reported that the murine lacrimal gland is capable of repair after experimentally induced injury and that the number of stem/progenitor cells was increased during the repair phase (2–3 days after injury). The aim of the present study was to determine whether these cells can be isolated from the lacrimal gland and propagated in vitro. Methods. Lacrimal gland injury was induced by injection of interleukin (IL)-1, and injection of saline vehicle served as control. Two and half days after injection, the lacrimal glands were removed and used to prepare explants or acinar cells for tissue culture. Cells derived from the explants and the acinar cells were grown in DMEM supplemented with 10% fetal bovine serum. Cells were stained for the stem cells markers, nestin, vimentin, ABCG2, and Sca-1. Cell proliferation was measured using an antibody against Ki67 or a cell-counting kit. The adipogenic capability of these cells was also tested in vitro. Results. Results show that nestin-positive cells can be isolated from IL-1–injected, but not saline-injected, lacrimal glands. A population of nestin-positive cells was also positive for vimentin, an intermediate filament protein expressed by mesenchymal cells. In addition, cultured cells expressed two other markers of stem cells, ABCG2 and Sca-1. These cells proliferated in vitro and can be induced to form adipocytes, attesting to their mesenchymal stem cell property. Conclusions. Murine lacrimal glands contain mesenchymal stem cells that seem to play a pivotal role in tissue repair. PMID:21178145

Characterization of the synthesis and expression of the GTA-kinase from transformed and normal rodent cells.

PubMed

Kerr, M; Fischer, J E; Purushotham, K R; Gao, D; Nakagawa, Y; Maeda, N; Ghanta, V; Hiramoto, R; Chegini, N; Humphreys-Beher, M G

1994-08-02

The murine transformed cell line YC-8 and beta-adrenergic receptor agonist (isoproternol) treated rat and mouse parotid gland acinar cells ectopically express cell surface beta 1-4 galactosyltransferase during active proliferation. This activity is dependent upon the expression of the GTA-kinase (p58) in these cells. Using total RNA, cDNA clones for the protein coding region of the kinase were isolated by reverse transcriptase-PCR cloning. DNA sequence analysis failed to show sequence differences with the normal homolog from mouse cells although Southern blot analysis of YC-8, and a second cell line KI81, indicated changes in the restriction enzyme digestion profile relative to murine cell lines which do not express cell surface galactosyltransferase. The rat cDNA clone from isoproterenol-treated salivary glands showed a high degree of protein and nucleic acid sequence homology to the GTA-kinase from both murine and human sources. Northern blot analysis of YC-8 and a control cell line LSTRA revealed the synthesis of a major 3.0 kb mRNA from both cell lines plus the unique expression of a 4.5 kb mRNA in the YC-8 cells. Reverse transcriptase-PCR of LSTRA and YC-8 confirmed the increased steady state levels of the GTA-kinase mRNA in YC-8. In the mouse, induction of cell proliferation by isoproterenol resulted in a 50-fold increase in steady state mRNA levels for the kinase over the low level of expression in quiescent cells. Expression of the rat 3' untranslated region in rat parotid cells in vitro led to an increased rate of DNA synthesis, cell number an ectopic expression of cell surface galactosyltransferase in the sense orientation. Antisense expression or vector alone did not alter growth characteristics of acinar cells. A polyclonal antibody monospecific to a murine amino terminal peptide sequence revealed a uniform distribution of GTA-kinase over the cytoplasm of acinar and duct cells of control mouse parotid glands. However, upon growth stimulation, kinase was

Phagocytosis of antibody‐opsonized tumor cells leads to the formation of a discrete vacuolar compartment in macrophages

PubMed Central

Velmurugan, Ramraj; Ramakrishnan, Sreevidhya; Kim, Mingin

2018-01-01

Despite the rapidly expanding use of antibody‐based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody‐opsonized tumor cells is limited. Here we report the formation of a phagosome‐associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2‐positive cancer cells in the presence of the HER2‐specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome‐associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the destruction of phagocytosed, antibody‐opsonized cancer cells by macrophages. PMID:29437282

The Effector TepP Mediates Recruitment and Activation of Phosphoinositide 3-Kinase on Early Chlamydia trachomatis Vacuoles.

PubMed

Carpenter, Victoria; Chen, Yi-Shan; Dolat, Lee; Valdivia, Raphael H

2017-01-01

Chlamydia trachomatis delivers multiple type 3 secreted effector proteins to host epithelial cells to manipulate cytoskeletal functions, membrane dynamics, and signaling pathways. TepP is the most abundant effector protein secreted early in infection, but its molecular function is poorly understood. In this report, we provide evidence that TepP is important for bacterial replication in cervical epithelial cells, activation of type I IFN genes, and recruitment of class I phosphoinositide 3-kinases (PI3K) and signaling adaptor protein CrkL to nascent pathogen-containing vacuoles (inclusions). We also show that TepP is a target of tyrosine phosphorylation by Src kinases but that these modifications do not appear to influence the recruitment of PI3K or CrkL. The translocation of TepP correlated with an increase in the intracellular pools of phosphoinositide-(3,4,5)-triphosphate but not the activation of the prosurvival kinase Akt, suggesting that TepP-mediated activation of PI3K is spatially restricted to early inclusions. Furthermore, we linked PI3K activity to the dampening of transcription of type I interferon (IFN)-induced genes early in infection. Overall, these findings indicate that TepP can modulate cell signaling and, potentially, membrane trafficking events by spatially restricted activation of PI3K. IMPORTANCE This article shows that Chlamydia recruits PI3K, an enzyme important for host cell survival and internal membrane functions, to the pathogens inside cells by secreting a scaffolding protein called TepP. TepP enhances Chlamydia replication and dampens the activation of immune responses.

Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.

PubMed

Taylor, M J; Baicu, S

2011-11-01

A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment. As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method

Methods for assessing autophagy and autophagic cell death.

PubMed

Tasdemir, Ezgi; Galluzzi, Lorenzo; Maiuri, M Chiara; Criollo, Alfredo; Vitale, Ilio; Hangen, Emilie; Modjtahedi, Nazanine; Kroemer, Guido

2008-01-01

Autophagic (or type 2) cell death is characterized by the massive accumulation of autophagic vacuoles (autophagosomes) in the cytoplasm of cells that lack signs of apoptosis (type 1 cell death). Here we detail and critically assess a series of methods to promote and inhibit autophagy via pharmacological and genetic manipulations. We also review the techniques currently available to detect autophagy, including transmission electron microscopy, half-life assessments of long-lived proteins, detection of LC3 maturation/aggregation, fluorescence microscopy, and colocalization of mitochondrion- or endoplasmic reticulum-specific markers with lysosomal proteins. Massive autophagic vacuolization may cause cellular stress and represent a frustrated attempt of adaptation. In this case, cell death occurs with (or in spite of) autophagy. When cell death occurs through autophagy, on the contrary, the inhibition of the autophagic process should prevent cellular demise. Accordingly, we describe a strategy for discriminating cell death with autophagy from cell death through autophagy.

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

PubMed

Nishihara, Eri; Yokota, Etsuo; Tazaki, Akira; Orii, Hidefumi; Katsuhara, Maki; Kataoka, Kensuke; Igarashi, Hisako; Moriyama, Yoshinori; Shimmen, Teruo; Sonobe, Seiji

2008-03-01

The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.

Capsaicin-Sensitive Sensory Nerves Are Necessary for the Protective Effect of Ghrelin in Cerulein-Induced Acute Pancreatitis in Rats

PubMed Central

Bonior, Joanna; Warzecha, Zygmunt; Ceranowicz, Piotr; Gajdosz, Ryszard; Pierzchalski, Piotr; Kot, Michalina; Leja-Szpak, Anna; Nawrot-Porąbka, Katarzyna; Link-Lenczowski, Paweł; Olszanecki, Rafał; Bartuś, Krzysztof; Trąbka, Rafał; Kuśnierz-Cabala, Beata; Dembiński, Artur; Jaworek, Jolanta

2017-01-01

Ghrelin was shown to exhibit protective and therapeutic effect in the gut. Aim of the study was to investigate the role of sensory nerves (SN) in the protective effect of ghrelin in acute pancreatitis (AP). Studies were performed on male Wistar rats or isolated pancreatic acinar cells. After capsaicin deactivation of sensory nerves (CDSN) or treatment with saline, rats were pretreated intraperitoneally with ghrelin or saline. In those rats, AP was induced by cerulein or pancreases were used for isolation of pancreatic acinar cells. Pancreatic acinar cells were incubated in cerulein-free or cerulein containing solution. In rats with intact SN, pretreatment with ghrelin led to a reversal of the cerulein-induced increase in pancreatic weight, plasma activity of lipase and plasma concentration of tumor necrosis factor-α (TNF-α). These effects were associated with an increase in plasma interleukin-4 concentration and reduction in histological signs of pancreatic damage. CDSN tended to increase the severity of AP and abolished the protective effect of ghrelin. Exposure of pancreatic acinar cells to cerulein led to increase in cellular expression of mRNA for TNF-α and cellular synthesis of this cytokine. Pretreatment with ghrelin reduced this alteration, but this effect was only observed in acinar cells obtained from rats with intact SN. Moreover, CDSN inhibited the cerulein- and ghrelin-induced increase in gene expression and synthesis of heat shock protein 70 (HSP70) in those cells. Ghrelin exhibits the protective effect in cerulein-induced AP on the organ and pancreatic acinar cell level. Sensory nerves ablation abolishes this effect. PMID:28665321

Pollen tube reuses intracellular components of nucellar cells undergoing programmed cell death in Pinus densiflora.

PubMed

Hiratsuka, Rie; Terasaka, Osamu

2011-04-01

Through the process known as programmed cell death (PCD), nucelli of Pinus densiflora serve as the transmitting tissue for growth of the pollen tube. We sought to clarify the processes of degradation of nucellar cell components and their transport to the pollen tube during PCD in response to pollen tube penetration of such nucelli. Stimulated by pollination, synthesis of large amounts of starch grains occurred in cells in a wide region of the nucellus, but as the pollen tube penetrated the nucellus, starch grains were degraded in amyloplasts of nucellar cells. In cells undergoing PCD, electron-dense vacuoles with high membrane contrast appeared, assumed a variety of autophagic structures, expanded, and ultimately collapsed and disappeared. Vesicles and electron-dense amorphous materials were released inside the thickened walls of cells undergoing PCD, and those vesicles and materials reaching the pollen tube after passing through the extracellular matrix were taken into the tube by endocytosis. These results show that in PCD of nucellar cells, intracellular materials are degraded in amyloplasts and vacuoles, and some of the degraded material is supplied to the pollen tube by vesicular transport to support tube growth.

Efflux of a range of antimalarial drugs and 'chloroquine resistance reversers' from the digestive vacuole in malaria parasites with mutant PfCRT.

PubMed

Lehane, Adele M; Kirk, Kiaran

2010-08-01

Chloroquine-resistant malaria parasites (Plasmodium falciparum) show an increased leak of H(+) ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H(+), out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasite's chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various 'chloroquine resistance reversers' induce an increased leak of H(+) from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild-type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound. © 2010 Blackwell Publishing Ltd.

A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi.

PubMed

Montalvetti, Andrea; Rohloff, Peter; Docampo, Roberto

2004-09-10

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3). glycerol transport was detected. No Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae.

PubMed

Sekito, Takayuki; Chardwiriyapreecha, Soracom; Sugimoto, Naoko; Ishimoto, Masaya; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2014-01-01

Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

New aspects of membrane dynamics of Amoeba proteus contractile vacuole revealed by vital staining with FM 4-64.

PubMed

Nishihara, E; Shimmen, T; Sonobe, S

2007-01-01

The contractile vacuole (CV) cycle of Amoeba proteus has been studied by phase contrast and electron microscopy. However, the understanding of membrane dynamics in this cycle is still poor. In this study, we used live imaging by fluorescence microscopy to obtain new insights. We succeeded in staining the CV with a styryl dye, FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide), and obtained the following results. (1) The CV membrane was directly stained with the dye in the external medium when the CV pore opened upon contraction. This indicates that transfer of plasma membrane to the CV does not occur. (2) The membrane dynamics during the CV cycle were elucidated. In particular, the fluorescent CV membrane was maintained as an aggregate just after contraction and the vacuole re-formed from the aggregate. Staining was maintained during continued contraction cycles. We conclude that the CV membrane is maintained during the CV cycle.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells.

PubMed

Perry, Clint; Quissell, David O; Reyland, Mary E; Grichtchenko, Irina I

2008-11-01

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.

Traffic to the malaria parasite food vacuole: a novel pathway involving a phosphatidylinositol 3-phosphate-binding protein.

PubMed

McIntosh, Michael T; Vaid, Ankush; Hosgood, H Dean; Vijay, Justin; Bhattacharya, Anindita; Sahani, Mayurbhai H; Baevova, Pavlina; Joiner, Keith A; Sharma, Pushkar

2007-04-13

Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV.

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

PubMed

Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

2008-12-01

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

Distribution and developmental changes of ghrelin-immunopositive cells in the pancreas of African ostrich chicks (Struthio camelus).

PubMed

Wang, J X; Li, P; Zhang, X T; Ye, L X

2017-09-01

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by multiple cell types and affects feeding behavior, metabolic regulation, and energy balance. In the mammalian pancreas, the types of endocrine cells that are immunoreactive to ghrelin vary. However, little was known about its distribution and developmental changes in the pancreas of African ostrich chicks (Struthio camelus). In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-immunopositive (ghrelin-ip) cells in the pancreas of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-ip cells were found in both the pancreatic islets and acinar cell regions. The greatest number of ghrelin-ip cells were found in the pancreatic islets, and were primarily observed at the periphery of the islets; some ghrelin-ip cells were also located in the central portion of the pancreatic islets. Interestingly, from postnatal d 1 to d 90, there was a steady decrease in the number of ghrelin-ip cells in the pancreatic islets and acinar cell regions. These results clearly demonstrated that ghrelin-ip cells exist and decreased with age in the African ostrich pancreas from postnatal d 1 to d90. Thus, these findings indicated that ghrelin may be involved in the development of the pancreas in the African ostrich. © 2017 Poultry Science Association Inc.

Imaging flow cytometry for the screening of compounds that disrupt the Plasmodium falciparum digestive vacuole.

PubMed

Chia, Wan Ni; Lee, Yan Quan; Tan, Kevin Shyong-Wei

2017-01-01

Malaria, despite being one of the world's oldest infectious diseases, remains difficult to eradicate because the parasite is rapidly developing resistance to frontline chemotherapies. Previous studies have shown that the parasite exhibits features resembling programmed cell death upon treatment with drugs that disrupt its digestive vacuole (DV), providing a phenotypic readout that can be detected using the imaging flow cytometer. Large compound collections can thus be screened to identify drugs that are able to disrupt the DV of the malaria parasite using this high-content high-throughput screening platform. As a proof-of-concept, 4440 compounds were screened using this platform in 4months and 254 hits (5.7% hit rate) were obtained. Additionally, 25 compounds (0.6% top hit rate) were observed to retain potent DV disruption activity that was comparable to the canonical DV disruptive drug chloroquine when tested at a ten-fold lower concentration from the original screen. This pilot study demonstrates the robustness and high-throughput capability of the imaging flow cytometer and we report herein the methodology of this screening assay. Copyright © 2016 Elsevier Inc. All rights reserved.

MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

PubMed

Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

2016-03-01

Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

Involvement of Ca2+ in Vacuole Degradation Caused by a Rapid Temperature Decrease in Saintpaulia Palisade Cells: A Case of Gene Expression Analysis in a Specialized Small Tissue.

PubMed

Ohnishi, Miwa; Kadohama, Noriaki; Suzuki, Yoshihiro; Kajiyama, Tomoharu; Shichijo, Chizuko; Ishizaki, Kimitsune; Fukaki, Hidehiro; Iida, Hidetoshi; Kambara, Hideki; Mimura, Tetsuro

2015-07-01

Saintpaulia (African violet) leaves are known to be damaged by a rapid temperature decrease when cold water is applied to the leaf surface; the injury is ascribed to the chloroplast damage caused by the cytosolic pH decrease following the degradation of the vacuolar membrane in the palisade cells. In this report, we present evidence for the involvement of Ca(2+) in facilitating the collapse of the vacuolar membrane and in turn in the temperature sensitivity of Saintpaulia leaves. In the presence of a Ca(2+) chelator (EGTA) or certain Ca(2+) channel inhibitors (Gd(3+) or La(3+)) but not others (verapamil or nifedipine), the pH of the vacuole, monitored through BCECF (2',7'-bis(carboxyethyl)-4 or 5-carboxyfluorescein) fluorescence, did not increase in response to a rapid temperature drop. These pharmacological observations are consistent with the involvement of mechanosensitive Ca(2+) channels in the collapse of the vacuolar membrane. The high level of expression of an MCA- (Arabidopsis mechanosensitive Ca(2+) channel) like gene, a likely candidate for a mechanosensitive Ca(2+) channel(s) in plant cells, was confirmed in the palisade tissue in Saintpaulia leaves by using a newly developed method of gene expression analysis for the specialized small tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Co-localization of endogenous Arf6 and its activator EFA6D in the granular convoluted tubule cells of mouse submandibular glands under normal conditions and when stimulated by isoproterenol, noradrenaline and carbachol.

PubMed

Tachow, Apussara; Thoungseabyoun, Wipawee; Phuapittayalert, Laorrat; Petcharat, Kanoktip; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

2017-10-01

This study proposed to investigate the localization at light and electron microscopic levels of Arf6 and its activator EFA6D in the mouse submandibular gland (SMG) under normal conditions and when stimulated by adrenergic or cholinergic agonists. SMGs of male adult mice were utilized for immunoblotting and immuno-light and -electron microscopic analyses. Isoproterenol and noradrenalin were used as adrenergics, while carbachol was used for the cholinergic stimulant. SMGs were examined at 15, 30, 60 and 120min after intraperitoneal injection of these agents. Immunoreactivities for both Arf6 and its activator EFA6D were similarly intense in the basolateral domain of GCTs, but no significant immunoreactivities were seen in the apical domain of GCT cells or any domain of acinar cells under normal conditions. In immuno-electron microscopy, the immunoreactive materials were mainly deposited on the basolateral plasma membranes and subjacent cytoplasm. Shortly after injection of isoproterenol and noradrenaline, but not carbachol, the immunoreactivities for both molecules were additionally seen on the apical plasmalemma of most, if not all, GCT cells, but not acinar cells. The present findings suggest that the direct involvement of Arf6/EFA6D in regulatory exocytosis at the apical plasma membrane of acinar and GCT cells is apparently to be smaller, if present, than that of endocytosis at the basolateral membranes of GCT cells under normal conditions. This also suggests that the two molecules function additionally at the apical membrane of GCT cells for modulation of saliva secretion under β-adrenoceptor stimulation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Live cell imaging of phosphoinositide dynamics during Legionella infection.

PubMed

Weber, Stephen; Hilbi, Hubert

2014-01-01

The "accidental" pathogen Legionella pneumophila replicates intracellularly in a distinct compartment, the Legionella-containing vacuole (LCV). To form this specific pathogen vacuole, the bacteria translocate via the Icm/Dot type IV secretion system approximately 300 different effector proteins into the host cell. Several of these secreted effectors anchor to the cytoplasmic face of the LCV membrane by binding to phosphoinositide (PI) lipids. L. pneumophila thus largely controls the localization of secreted bacterial effectors and the recruitment of host factors to the LCV through the modulation of the vacuole membrane PI pattern. The LCV PI pattern and its dynamics can be studied in real-time using fluorescently labeled protein probes stably produced by the soil amoeba Dictyostelium discoideum. In this chapter, we describe a protocol to (1) construct and handle amoeba model systems as a tool for observing PIs in live cell imaging, (2) capture rapid changes in membrane PI patterning during uptake events, and (3) observe the dynamics of LCV PIs over the course of a Legionella infection.

Creating new β cells: cellular transmutation by genomic alchemy.

PubMed

Moss, Larry G

2013-03-01

To address insulin insufficiency, diabetes research has long focused on techniques for replacing insulin-producing β cells. Studies in mice have suggested that, under some conditions, α cells possess the capacity to transdifferentiate into β cells, although the mechanisms that drive this conversion are unclear. In this issue, Bramswig et al. analyzed the methylation states of purified human α, β, and acinar cells and found α cells exhibit intrinsic phenotypic plasticity associated with specific histone methylation profiles. In addition to expanding our understanding of this potential source of β cells, this compendium of carefully generated human gene expression and epigenomic data in islet cell subtypes constitutes a truly valuable resource for the field.

Creating new β cells: cellular transmutation by genomic alchemy

PubMed Central

Moss, Larry G.

2013-01-01

To address insulin insufficiency, diabetes research has long focused on techniques for replacing insulin-producing β cells. Studies in mice have suggested that, under some conditions, α cells possess the capacity to transdifferentiate into β cells, although the mechanisms that drive this conversion are unclear. In this issue, Bramswig et al. analyzed the methylation states of purified human α, β, and acinar cells and found α cells exhibit intrinsic phenotypic plasticity associated with specific histone methylation profiles. In addition to expanding our understanding of this potential source of β cells, this compendium of carefully generated human gene expression and epigenomic data in islet cell subtypes constitutes a truly valuable resource for the field. PMID:23434598