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Sample records for acinar cell vacuole

  1. Acinar Cell Carcinoma of the Pancreas

    Institute of Scientific and Technical Information of China (English)

    Hua Li; Qiang Li

    2008-01-01

    Acinar cell carcinoma of the pancreas is a rare tumor which is defined as a carcinoma that exhibits pancreatic enzyme production by neoplastic cells. This review includes re-cent developments in our understanding of the epidemiology and pathogenesis of ACC, imaging and pathological diagnosis and ap-proaches to treatment with reference to the literature.

  2. Papillocystic Variant of Acinar Cell Pancreatic Carcinoma

    Directory of Open Access Journals (Sweden)

    Jasim Radhi

    2010-01-01

    Full Text Available Acinar cell pancreatic carcinoma is a rare solid malignant neoplasm. Recent review of the literature showed occasional cases with papillary or papillocystic growth patterns, ranging from 2 to 5 cm in diameter. We report a large 10 cm pancreatic tumor with papillocystic pathology features involving the pancreatic head. The growth pattern of these tumors could be mistaken for intraductal papillary mucinous tumors or other pancreatic cystic neoplasms.

  3. Inflammatory role of the acinar cells during acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Isabel; De; Dios

    2010-01-01

    Pancreatic acinar cells are secretory cells whose main function is to synthesize, store and f inally release digestive enzymes into the duodenum. However, in response to noxious stimuli, acinar cells behave like real inflammatory cells because of their ability to activate signalling transduction pathways involved in the expression of inflammatory mediators. Mediated by the kinase cascade, activation of Nuclear factor-κB, Activating factor-1 and Signal transducers and activators of transcription transcription factors has been demonstrated in acinar cells, resulting in overexpression of inflammatory genes. In turn, kinase activity is down-regulated by protein phosphatases and the f inal balance between kinase and phosphatase activity will determine the capability of the acinar cells to produce inflammatory factors. The kinase/ phosphatase pair is a redox-sensitive system in which kinase activation overwhelms phosphatase activity under oxidant conditions. Thus, the oxidative stress developed within acinar cells at early stages of acute pancreatitis triggers the activation of signalling pathways involved in the up-regulation of cytokines, chemokines and adhesion molecules. In this way, acinar cells trigger the release of the f irst inflammatory signals which can mediate the activation and recruitment of circulating inflammatorycells into the injured pancreas. Accordingly, the role of acinar cells as promoters of the inflammatory response in acute pancreatitis may be considered. This concept leads to amplifying the focus from leukocyte to acinar cells themselves, to explain the local inflammation in early pancreatitis.

  4. Acinar Cell Cyst adenoma (Acinar Cystic Transformation) of the Pancreas: the Radiologic-Pathologic Features

    Energy Technology Data Exchange (ETDEWEB)

    Gumus, Mehmet; Algin, Oktay; Gundogdu, Haldun [Ataturk Training and Research Hospital, Ankara (Turkmenistan); Ugras, Serdar [Selcuk University, Selcuklu Medical Faculty, Konya (Turkmenistan)

    2011-02-15

    Acinar cystic transformation of the pancreas is also known as acinar cell cystadenoma (ACC), and this is an extremely rare benign lesion that was first described in April 2002. We report here on a case of a previously asymptomatic patient with pancreatic ACC and this was diagnosed by computed tomography (CT) and magnetic resonance imaging (MRI). To the best of our knowledge, there is no previous report concerning the CT or MRI features of ACC in the medical literature. We present here the CT, MRI and pathological findings of pancreatic ACC

  5. TGF-β1 promotes acinar to ductal metaplasia of human pancreatic acinar cells.

    Science.gov (United States)

    Liu, Jun; Akanuma, Naoki; Liu, Chengyang; Naji, Ali; Halff, Glenn A; Washburn, William K; Sun, Luzhe; Wang, Pei

    2016-01-01

    Animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for pancreatic ductal adenocarcinoma (PDAC) initiation. However, there has not been an adequate system to explore the mechanisms of human ADM induction. We have developed a flow cytometry-based, high resolution lineage tracing method and 3D culture system to analyse ADM in human cells. In this system, well-known mouse ADM inducers did not promote ADM in human cells. In contrast, TGF-β1 efficiently converted human acinar cells to duct-like cells (AD) in a SMAD-dependent manner, highlighting fundamental differences between the species. Functionally, AD cells gained transient proliferative capacity. Furthermore, oncogenic KRAS did not induce acinar cell proliferation, but did sustain the proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases. PMID:27485764

  6. Nitric oxide-induced signalling in rat lacrimal acinar cells

    DEFF Research Database (Denmark)

    Looms, Dagnia Karen; Tritsaris, K.; Dissing, S.

    2002-01-01

    The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO productio...... not by itself causing fast transient increases in [Ca2+]i. In addition, we suggest that endogenously produced NO activated by ß-adrenergic receptor stimulation, plays an important role in signalling to the surrounding tissue.......The aim of the present study was to investigate the physiological role of nitric oxide (NO) in mediating secretory processes in rat lacrimal acinar cells. In addition, we wanted to determine whether the acinar cells possess endogenous nitric oxide synthase (NOS) activity by measuring NO production......-adrenergic stimulation and not by a rise in [Ca2+]i alone.   We show that in rat lacrimal acinar cells, NO and cGMP induce Ca2+ release from intracellular stores via G kinase activation. However, the changes in [Ca2+]i are relatively small, suggesting that this pathway plays a modulatory role in Ca2+ signalling, thus...

  7. Calcium signaling of pancreatic acinar cells in the pathogenesis of pancreatitis.

    Science.gov (United States)

    Li, Jun; Zhou, Rui; Zhang, Jian; Li, Zong-Fang

    2014-11-21

    Pancreatitis is an increasingly common and sometimes severe disease that lacks a specific therapy. The pathogenesis of pancreatitis is still not well understood. Calcium (Ca(2+)) is a versatile carrier of signals regulating many aspects of cellular activity and plays a central role in controlling digestive enzyme secretion in pancreatic acinar cells. Ca(2+) overload is a key early event and is crucial in the pathogenesis of many diseases. In pancreatic acinar cells, pathological Ca(2+) signaling (stimulated by bile, alcohol metabolites and other causes) is a key contributor to the initiation of cell injury due to prolonged and global Ca(2+) elevation that results in trypsin activation, vacuolization and necrosis, all of which are crucial in the development of pancreatitis. Increased release of Ca(2+) from stores in the intracellular endoplasmic reticulum and/or increased Ca(2+) entry through the plasma membrane are causes of such cell damage. Failed mitochondrial adenosine triphosphate (ATP) production reduces re-uptake and extrusion of Ca(2+) by the sarco/endoplasmic reticulum Ca(2+)-activated ATPase and plasma membrane Ca(2+)-ATPase pumps, which contribute to Ca(2+) overload. Current findings have provided further insight into the roles and mechanisms of abnormal pancreatic acinar Ca(2+) signals in pancreatitis. The lack of available specific treatments is therefore an objective of ongoing research. Research is currently underway to establish the mechanisms and interactions of Ca(2+) signals in the pathogenesis of pancreatitis.

  8. Ca2+ signaling in pancreatic acinar cells: physiology and pathophysiology

    Directory of Open Access Journals (Sweden)

    O.H. Petersen

    2009-01-01

    Full Text Available The pancreatic acinar cell is a classical model for studies of secretion and signal transduction mechanisms. Because of the extensive endoplasmic reticulum and the large granular compartment, it has been possible - by direct measurements - to obtain considerable insights into intracellular Ca2+ handling under both normal and pathological conditions. Recent studies have also revealed important characteristics of stimulus-secretion coupling mechanisms in isolated human pancreatic acinar cells. The acinar cells are potentially dangerous because of the high intra-granular concentration of proteases, which become inappropriately activated in the human disease acute pancreatitis. This disease is due to toxic Ca2+ signals generated by excessive liberation of Ca2+ from both the endoplasmic reticulum and the secretory granules.

  9. Effects of Benzodiazepines on Acinar and Myoepithelial Cells

    Science.gov (United States)

    Mattioli, Tatiana M. F.; Alanis, Luciana R. A.; Sapelli, Silvana da Silva; de Lima, Antonio A. S.; de Noronha, Lucia; Rosa, Edvaldo A. R.; Althobaiti, Yusuf S.; Almalki, Atiah H.; Sari, Youssef; Ignacio, Sergio A.; Johann, Aline C. B. R.; Gregio, Ana M. T.

    2016-01-01

    Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells. Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated. Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60). Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands. PMID:27445812

  10. Effects of Benzodiazepines on Acinar and Myoepithelial Cells

    Science.gov (United States)

    Mattioli, Tatiana M. F.; Alanis, Luciana R. A.; Sapelli, Silvana da Silva; de Lima, Antonio A. S.; de Noronha, Lucia; Rosa, Edvaldo A. R.; Althobaiti, Yusuf S.; Almalki, Atiah H.; Sari, Youssef; Ignacio, Sergio A.; Johann, Aline C. B. R.; Gregio, Ana M. T.

    2016-01-01

    Background: Benzodiazepines (BZDs), the most commonly prescribed psychotropic drugs with anxiolytic action, may cause hyposalivation. It has been previously shown that BZDs can cause hypertrophy and decrease the acini cell number. In this study, we investigated the effects of BZDs and pilocarpine on rat parotid glands, specifically on acinar, ductal, and myoepithelial cells. Methods: Ninety male Wistar rats were divided into nine groups. Control groups received a saline solution for 30 days (C30) and 60 days (C60), and pilocarpine (PILO) for 60 days. Experimental groups received lorazepam (L30) and midazolam (M30) for 30 days. Another group (LS60 or MS60) received lorazepam or midazolam for 30 days, respectively, and saline for additional 30 days. Finally, other groups (LP60 or MP60) received either lorazepam or midazolam for 30 days, respectively, and pilocarpine for additional 30 days. The expression of calponin in myoepithelial cells and the proliferating cell nuclear antigen (PCNA) in acinar and ductal cells were evaluated. Results: Animals treated with lorazepam showed an increase in the number of positive staining cells for calponin as compared to control animals (p < 0.05). Midazolam administered with pilocarpine (MP60) induced an increase in the proliferation of acinar and ductal cells and a decrease in the positive staining cells for calponin as compared to midazolam administered with saline (MS60). Conclusion: We found that myoepithelial cells might be more sensitive to the effects of BZD than acinar and ductal cells in rat parotid glands.

  11. File list: DNS.Pan.20.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available DNS.Pan.20.AllAg.Pancreatic_acinar_cells mm9 DNase-seq Pancreas Pancreatic acinar c...ells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/DNS.Pan.20.AllAg.Pancreatic_acinar_cells.bed ...

  12. File list: ALL.Pan.05.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Pan.05.AllAg.Pancreatic_acinar_cells mm9 All antigens Pancreas Pancreatic acina...r cells SRX327163,SRX327162,SRX327160,SRX327161 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Pan.05.AllAg.Pancreatic_acinar_cells.bed ...

  13. File list: Oth.Pan.05.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Pan.05.AllAg.Pancreatic_acinar_cells mm9 TFs and others Pancreas Pancreatic aci...nar cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Pan.05.AllAg.Pancreatic_acinar_cells.bed ...

  14. File list: ALL.Pan.50.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available ALL.Pan.50.AllAg.Pancreatic_acinar_cells mm9 All antigens Pancreas Pancreatic acina...r cells SRX327161,SRX327160,SRX327162,SRX327163 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/ALL.Pan.50.AllAg.Pancreatic_acinar_cells.bed ...

  15. File list: Pol.Pan.50.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.50.AllAg.Pancreatic_acinar_cells mm9 RNA polymerase Pancreas Pancreatic aci...nar cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Pan.50.AllAg.Pancreatic_acinar_cells.bed ...

  16. File list: Pol.Pan.10.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Pol.Pan.10.AllAg.Pancreatic_acinar_cells mm9 RNA polymerase Pancreas Pancreatic aci...nar cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Pol.Pan.10.AllAg.Pancreatic_acinar_cells.bed ...

  17. Salivary gland homeostasis is maintained through acinar cell self-duplication.

    Science.gov (United States)

    Aure, Marit H; Konieczny, Stephen F; Ovitt, Catherine E

    2015-04-20

    Current dogma suggests that salivary gland homeostasis is stem cell dependent. However, the extent of stem cell contribution to salivary gland maintenance has not been determined. We investigated acinar cell replacement during homeostasis, growth, and regeneration, using an inducible CreER(T2) expressed under the control of the Mist1 gene locus. Genetic labeling, followed by a chase period, showed that acinar cell replacement is not driven by the differentiation of unlabeled stem cells. Analysis using R26(Brainbow2.1) reporter revealed continued proliferation and clonal expansion of terminally differentiated acinar cells in all major salivary glands. Induced injury also demonstrated the regenerative potential of pre-labeled acinar cells. Our results support a revised model for salivary gland homeostasis based predominantly on self-duplication of acinar cells, rather than on differentiation of stem cells. The proliferative capacity of differentiated acinar cells may prove critical in the implementation of cell-based strategies to restore the salivary glands.

  18. Adrenoceptor-activated nitric oxide synthesis in salivary acinar cells

    DEFF Research Database (Denmark)

    Looms, Dagnia; Dissing, Steen; Tritsaris, Katerina;

    2000-01-01

    We investigated the cellular regulation of nitric oxide synthase (NOS) activity in isolated acinar cells from rat parotid and human labial salivary glands, using the newly developed fluorescent nitric oxide (NO) indicator, DAF-2. We found that sympathetic stimulation with norepinephrine (NE) caused...... a strong increase in NO synthesis that was not seen after parasympathetic stimulation with acetylcholine. In rat parotid acinar cells, we furthermore investigated to which extent the NOS activity was dependent on the intracellular free Ca2+ concentration ([Ca2+]i) by simultaneously measuring NO synthesis...... not cause significant NO synthesis. We furthermore found that activating adrenoceptors with NE causes synthesis of cGMP by activating a guanylyl cyclase, and that an enhanced [cGMP] evoked by use of caged cGMP causes Ca2+ release from internal stores. Thus, upon sympathetic stimulation, salivary gland acini...

  19. Ultrastructural morphometry of parotid acinar cells following fractionated irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Grehn, A.-L.; Gustafsson, H.; Franzen, L.; Thornell, L.-E.; Henriksson, R. [Umeaa Univ. (Sweden)

    1997-01-01

    The aim of this study was to evaluate the long term effects on the ultrastructure of parotid glands after fractionated irradiation. The method implemented involved 5 x 6 Gy and 5 x 8 Gy, Monday to Friday 6 MV photons. By unilateral irradiation, the contralateral parotid gland served as a control. Although irradiation diminished the acinar cell density in light microscopic sections from 75 to 32% after 5 x 8 Gy of irradiation, ultrastructural morphometry could not detect any statistically significant differences in acinar cell size, nuclear size, nuclear density, granule area, mean granule size, or granule density. In general, greater differences were seen between rats receiving 30 or 40 Gy, on both the irradiated and the control side, than between the irradiated side and the control side. This was interpreted as due to differences in the nutritional state of the animals. This analysis concluded that individual acinar cells that survive irradiation seem not to be damaged in the long term when evaluated at the ultrastructural level. The study further stresses the importance of adequate sampling sizes and the use of adequate controls. (author).

  20. Nicotine as a mitogenic stimulus for pancreatic acinar cell proliferation

    Institute of Scientific and Technical Information of China (English)

    Parimal Chowdhury; Kodetthoor B Udupa

    2006-01-01

    Cell proliferation is an important process in life for growth of normal and cancer cells. The signal transduction pathways activated during this process are strictly regulated. This editorial focuses on the role of nicotine,a mitogen, in the induction of signaling pathways resulting in proliferation of pancreatic tumor cells and compares these events with those in normal acinar cells isolated from the rat pancreas. The data shows striking similarities between these two cellular systems.In addition, the editorial reviews very recent literature of the contribution of MAPK signaling in cell lines associated with human diseases. A prospective cellular model of nicotine induced activation of MAPK cascade is presented.

  1. Recurrent Pancreatitis Due to a Cystic Pancreatic Tumor: A Rare Presentation of Acinar Cell Carcinoma

    OpenAIRE

    Raimondo M; Krishna M; Nguyen J; Scolapio J; Aqel B

    2004-01-01

    CONTEXT: Acinar cell carcinoma is an uncommon malignancy of the pancreas. It has characteristic histomorphology, immunohistochemistry profile, and clinicopathological behavior. CASE REPORT: We report a rare case of recurrent pancreatitis secondary to acinar cell carcinoma of the pancreas. We describe the endoscopic ultrasound characteristic, treatment and the surgical outcome. CONCLUSIONS: Acinar cell carcinoma should be considered in the differential diagnosis of cystic pancreatic tumors pre...

  2. Recurrent Pancreatitis Due to a Cystic Pancreatic Tumor: A Rare Presentation of Acinar Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Raimondo M

    2004-05-01

    Full Text Available CONTEXT: Acinar cell carcinoma is an uncommon malignancy of the pancreas. It has characteristic histomorphology, immunohistochemistry profile, and clinicopathological behavior. CASE REPORT: We report a rare case of recurrent pancreatitis secondary to acinar cell carcinoma of the pancreas. We describe the endoscopic ultrasound characteristic, treatment and the surgical outcome. CONCLUSIONS: Acinar cell carcinoma should be considered in the differential diagnosis of cystic pancreatic tumors presenting with recurrent pancreatitis.

  3. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/106 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]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',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition 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 Ni to inhibit adenylate cyclase

  4. Pancreatic acinar cells: molecular insight from studies of signal-transduction using transgenic animals.

    Science.gov (United States)

    Yule, David I

    2010-11-01

    Pancreatic acinar cells are classical exocrine gland cells. The apical regions of clusters of coupled acinar cells collectively form a lumen which constitutes the blind end of a tube created by ductal cells - a structure reminiscent of a "bunch of grapes". When activated by neural or hormonal secretagogues, pancreatic acinar cells are stimulated to secrete a variety of proteins. These proteins are predominately inactive digestive enzyme precursors called "zymogens". Acinar cell secretion is absolutely dependent on secretagogue-induced increases in intracellular free Ca(2+). The increase in [Ca(2+)](i) has precise temporal and spatial characteristics as a result of the exquisite regulation of the proteins responsible for Ca(2+) release, Ca(2+) influx and Ca(2+) clearance in the acinar cell. This brief review discusses recent studies in which transgenic animal models have been utilized to define in molecular detail the components of the Ca(2+) signaling machinery which contribute to these characteristics.

  5. Acinar Cell Carcinoma of the Pancreas with Colon Involvement

    Directory of Open Access Journals (Sweden)

    Naoki Asayama

    2014-01-01

    Full Text Available We report a case of acinar cell carcinoma of the pancreas with colon involvement that was difficult to distinguish from primary colon cancer. A 60-year-old man was admitted with a 1-month history of diarrhea. Contrast-enhanced computed tomography (CT revealed a large tumor (10.6×11.6 cm at the splenic flexure of the colon. Colonoscopy showed completely round ulcerative lesions, and biopsy revealed poorly differentiated adenocarcinoma. Left hemicolectomy, resection of the jejunum and pancreas body and tail, and splenectomy were performed based on a diagnosis of descending colon cancer (cT4N0M0, stage IIB, and surgery was considered to be curative. Diagnosis was subsequently confirmed as moderately differentiated acinar cell carcinoma of the pancreas by immunohistochemical staining (pT3N0M0, stage IIA. Multiple liver metastases with portal thrombosis were found 8 weeks postoperatively. Despite combination chemotherapy with oral S-1 and gemcitabine, the patient died of hepatic failure with no effect of chemotherapy 14 weeks postoperatively. Correct diagnosis was difficult to determine preoperatively from the clinical, CT, and colonoscopy findings. Moreover, the disease was extremely aggressive even after curative resection. Physicians should consider pancreatic cancer in the differential diagnosis of similar cases.

  6. File list: NoD.Pan.20.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available NoD.Pan.20.AllAg.Pancreatic_acinar_cells mm9 No description Pancreas Pancreatic aci...nar cells http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/NoD.Pan.20.AllAg.Pancreatic_acinar_cells.bed ...

  7. File list: InP.Pan.10.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Pan.10.AllAg.Pancreatic_acinar_cells mm9 Input control Pancreas Pancreatic acin...ar cells SRX327162,SRX327163 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Pan.10.AllAg.Pancreatic_acinar_cells.bed ...

  8. File list: InP.Pan.50.AllAg.Pancreatic_acinar_cells [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available InP.Pan.50.AllAg.Pancreatic_acinar_cells mm9 Input control Pancreas Pancreatic acin...ar cells SRX327162,SRX327163 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/InP.Pan.50.AllAg.Pancreatic_acinar_cells.bed ...

  9. Effects of hypothyroidism on the ultrastructure of rat pancreatic acinar cells: a stereological analysis

    OpenAIRE

    Blanco-Molina, A.; González-Reyes, J. A.; Torre-Cisneros, J; López-Miranda, J.; Nicolás, M.; Pérez-Jiménez, F.

    1991-01-01

    The morphological and stereological characteristics of the exocrine pancreas subcellular organelles from healthy and thyroidectomized rats have been studied. The acinar tissue from hypothyroid rats showed an interstitial edema and evidence of degenerative processes. Stereological parameters of zymogen granules were significantly reduced in thyroidectomized rats. The hypothyroidism induced degenerative changes in the pancreatic acinar cells as well as a decr...

  10. KRAS Mutations in Canine and Feline Pancreatic Acinar Cell Carcinoma.

    Science.gov (United States)

    Crozier, C; Wood, G A; Foster, R A; Stasi, S; Liu, J H W; Bartlett, J M S; Coomber, B L; Sabine, V S

    2016-07-01

    Companion animals may serve as valuable models for studying human cancers. Although KRAS is the most commonly mutated gene in human ductal pancreatic cancers (57%), with mutations frequently occurring at codons 12, 13 and 61, human pancreatic acinar cell carcinomas (ACCs) lack activating KRAS mutations. In the present study, 32 pancreatic ACC samples obtained from 14 dogs and 18 cats, including seven metastases, were analyzed for six common activating KRAS mutations located in codons 12 (n = 5) and 13 (n = 1) using Sequenom MassARRAY. No KRAS mutations were found, suggesting that, similar to human pancreatic ACC, KRAS mutations do not play a critical role in feline or canine pancreatic ACC. Due to the similarity of the clinical disease in dogs and cats to that of man, this study confirms that companion animals offer potential as a suitable model for investigating this rare subtype of pancreatic carcinoma. PMID:27290644

  11. Acinar Cell Carcinoma of the Pancreas: A Possible Role of S-1 as Chemotherapy for Acinar Cell Carcinoma. A Case Report

    Directory of Open Access Journals (Sweden)

    Tameyoshi Yamamoto

    2012-01-01

    Full Text Available Context Acinar cell carcinoma of the pancreas is a rare malignancy, accounting for 1-2% of pancreatic exocrine malignancies. This rarity makes it difficult to standardize a protocol of treatment for acinar cell carcinoma. Case report A 71-year-old male without any particular past history was referred to our institute with abdominal distention and mild liver dysfunction. Computed tomography (CT revealed a cystic lesion with a diameter of 3.5 cm, which originated from the neck of pancreas and had solid nodules inside. Several nodules were demonstrated surrounding the cystic tumor. Laparotomy and histological study demonstrated peritoneal dissemination of acinar cell carcinoma. The patient was treated with S-1 monotherapy (80 mg/m2 for four weeks with a two-week interval as one cycle. After one cycle of S-1 monotherapy, CT demonstrated remarkable shrinkage of the main tumor and disappearance of the nodules on the peritoneum. The patient underwent a radical distal pancreatectomy. The patient was then treated with 16 cycles of S-1 monotherapy after the radical pancreatectomy and remains without any recurrence of the disease two years later. Conclusion Initially inoperable acinar cell carcinoma was treated by monotherapy using S-1, resulting in curative operation and two years disease free survival post operation. S-1 might be more effective on acinar cell carcinoma, rather than gemcitabine

  12. Regeneration of parotid acinar cells after high radiation doses. A morphological study in rat

    Energy Technology Data Exchange (ETDEWEB)

    Gustafsson, H. [Depts. of Oto-Rhino-Laryngology, Histology and Cell Biology, Umeaa Univ. (Sweden); Franzen, L. [Dept. of Oncology, Umeaa Univ. (Sweden); Henriksson, R. [Dept. of Oncology, Umeaa Univ. (Sweden)

    1995-12-31

    The acute and late effects of fractionated irradiation on rat parotid gland acinar cells were studied by light and electron microscopy. At 10 days after the last irradiation session (6 Gy or 9 Gy daily during five consecutive days) no effects were seen. At 180 days, minor loss of acini was detectable after a total dose of 30 Gy. After 45 Gy a massive acinar loss was seen at that time; the number of acini had diminished and minor duct-like structures and scattered amounts of fibrous stroma dominated the slides. The remaining acini were disorganized and usually larger compared with the control side and to non-irradiated animals. The acinar cells appeared larger than in the controls. The custs were better preserved but the intercalated ducts often seemed to be larger than normal. We suggest that this phenomenon indicates a remaining capacity of the parotid gland to regenerate acinar cells even after high radiation doses. (orig.).

  13. Formation of salivary acinar cell spheroids in vitro above a polyvinyl alcohol-coated surface.

    Science.gov (United States)

    Chen, Min-Huey; Chen, Yi-Jane; Liao, Chih-Chen; Chan, Yen-Hui; Lin, Chia-Yung; Chen, Rung-Shu; Young, Tai-Hong

    2009-09-15

    Tissue engineering of salivary glands offers the potential for future use in the treatment of patients with salivary hypofunction. Biocompatible materials that promote acinar cell aggregation and function in vitro are an essential part of salivary gland tissue engineering. In this study, rat parotid acinar cells assembled into three-dimensional aggregates above the polyvinyl alcohol (PVA)-coated surface. These aggregates developed compact acinar cell spheroids resembling in vivo physiological condition, which were different from the traditional monolayered morphology in vitro. Cells remained viable and with better functional activity in response to acetylcholine in the spheroids and could form monolayered acinar cells when they were reinoculated on tissue culture polystyrene wells. To interpret the phenomenon further, we proposed that the formation of acinar cell spheroids on the PVA is mediated by a balance between two competing forces: the interactions of cell-PVA and cell-cell. This study demonstrated the formation of functional cell spheroids above a PVA-coated surface may provide an in vitro system for investigating cell behaviors for tissue engineering of artificial salivary gland.

  14. Analysis and Optimization of Nutritional Set-up for Murine Pancreatic Acinar Cells.

    Directory of Open Access Journals (Sweden)

    Kurup S

    2002-01-01

    Full Text Available CONTEXT: Pancreatic acinar cell cultivation poses a serious problem due to limitations in the in vitro survival time despite variations of dissociation protocols, culture media and nutrient supplements. OBJECTIVE: To establish a long term culture of murine pancreatic acinar cells which retain their viability, monolayer formation and responsiveness to secretagogues. In order to investigate the mechanism of the short-life of acinar cells studied in vitro, we studied their survival under the influence of different supplements on nutrient media. INTERVENTIONS: Dissociated pancreatic acini were prepared from BALB/c mice pancreata by collagenase digestion supplemented with bovine serum albumin fraction V and soybean trypsin inhibitor. A nutrient set-up was designed for their long term survival in vitro. RESULTS: It was observed that mouse pancreatic acinar cells dissociated in presence of bovine serum albumin fraction V and soybean trypsin inhibitor result in 95% viability. Further cultivation of these acinar cells in Waymouth's MB 752/1 medium supplemented with 10% fetal calf serum (v/v, soybean trypsin inhibitor, bovine serum albumin, dexamethasone, and epidermal growth factor results in their survival for more than 6 days in culture with 85% viability, retention of the secretagogue responsiveness and formation of a monolayer without any extracellular matrix coating. CONCLUSIONS: Our study clearly demonstrates that the addition of soybean trypsin inhibitor to culture medium reduces zymogen granule fragility and acinar cell death, thus increasing their viability for sufficiently long periods. The present study offers an excellent, in vitro model for the investigation of exocrine dysfunction in response to acinar cell injury.

  15. A computer-based automated algorithm for assessing acinar cell loss after experimental pancreatitis.

    Directory of Open Access Journals (Sweden)

    John F Eisses

    Full Text Available The change in exocrine mass is an important parameter to follow in experimental models of pancreatic injury and regeneration. However, at present, the quantitative assessment of exocrine content by histology is tedious and operator-dependent, requiring manual assessment of acinar area on serial pancreatic sections. In this study, we utilized a novel computer-generated learning algorithm to construct an accurate and rapid method of quantifying acinar content. The algorithm works by learning differences in pixel characteristics from input examples provided by human experts. HE-stained pancreatic sections were obtained in mice recovering from a 2-day, hourly caerulein hyperstimulation model of experimental pancreatitis. For training data, a pathologist carefully outlined discrete regions of acinar and non-acinar tissue in 21 sections at various stages of pancreatic injury and recovery (termed the "ground truth". After the expert defined the ground truth, the computer was able to develop a prediction rule that was then applied to a unique set of high-resolution images in order to validate the process. For baseline, non-injured pancreatic sections, the software demonstrated close agreement with the ground truth in identifying baseline acinar tissue area with only a difference of 1% ± 0.05% (p = 0.21. Within regions of injured tissue, the software reported a difference of 2.5% ± 0.04% in acinar area compared with the pathologist (p = 0.47. Surprisingly, on detailed morphological examination, the discrepancy was primarily because the software outlined acini and excluded inter-acinar and luminal white space with greater precision. The findings suggest that the software will be of great potential benefit to both clinicians and researchers in quantifying pancreatic acinar cell flux in the injured and recovering pancreas.

  16. THE CHANGES OF PANCREATIC ACINAR CELL FUNCTION IN ACUTE NECROTIZING PANCREATITIS OF RATS

    Institute of Scientific and Technical Information of China (English)

    余枭; 韩天权; 汤耀卿; 雷若庆; 夏宗勤

    2000-01-01

    Objective To evaluate the changes of pancreatic acinar cell functions in the rats with acute necrotizing pancreatitis (ANP). Methods Seventy SD rats were randomized into two groups: experimental group (n=35) and control group (n=35). To prepare the experimental model, the retrograde injection of 5% sodium taurocholate into the pancreatic duct was used for inducing ANP. Radioactive tracing by L- 3H-phenylalanine and autoradiography were performed for scoring the differences of changes of amino acid uptake, enzyme-protein synthesis and output from acinar cells in rats between both groups. Results No changes were observed in amino acid uptake and enzyme-protein synthesis in rats with dotted and haemorrhagic necrotizing foci as compared with control group. However, accumulated zymogen granules in the interstitial of acinar cells were seen in the experimental group. Conclusion It indicates that in experimental ANP rats, the functions of acinar cells in both amino acid uptake and protein synthesis were essentially normal, but the pathway of enzyme output was affected into ectopic secretion through the bottom or lateral cellular membrane of pancreatic acinar cell.

  17. Alteration of chaperonin60 and pancreatic enzyme in pancreatic acinar cell under pathological condition

    Institute of Scientific and Technical Information of China (English)

    Yong-Yu Li; Moise Bendayan

    2005-01-01

    AIM: To investigate the changes of chaperonin60 (Cpn60)and pancreatic enzymes in pancreatic acinar cells, and to explore their roles in the development of experimental diabetes and acute pancreatitis (AP).METHODS: Two different pathological models were replicated in Sprague-Dawley rats: streptozotocininduced diabetes and sodium deoxycholate-induced AP. The contents of Cpn60 and pancreatic enzymes in different compartments of the acinar cells were measured by quantitative immunocytochemistry.RESULTS: The levels of Cpn60 significantly increased in diabetes, but decreased in AP, especially in the zymogen granules of the pancreatic acinar cells. The elevation of Cpn60 was accompanied with the increased levels of pancreatic lipase and chymotrypsinogen in diabetes.However, a decreased Cpn60 level was accompanied by high levels of lipase and chymotrypsinogen in AP.The amylase level was markedly reduced in both the pathological conditions.CONCLUSION: The equilibrium between Cpn60 and pancreatic enzymes in the acinar cells breaks in AP, and Cpn60 content decreases, suggesting an insufficient chaperone capacity. This may promote the aggregation and autoactivation of the premature enzymes in the pancreatic acinar cells and play roles in the development of AP.

  18. Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Ge [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wan, Rong [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Hu, Yanling [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Ni, Jianbo [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Yin, Guojian; Xing, Miao [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Shen, Jie [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Tang, Maochun [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Chen, Congying [Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); Fan, Yuting; Xiao, Wenqin; Zhao, Yan [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Wang, Xingpeng, E-mail: wangxingpeng@hotmail.com [Department of Gastroenterology, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai (China); Department of Gastroenterology, Shanghai First People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai (China); and others

    2014-01-31

    Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway.

  19. Effect of Taurine on Acinar Cell Apoptosis and Pancreatic Fibrosis in Dibutyltin Dichloride-induced Chronic Pancreatitis

    Directory of Open Access Journals (Sweden)

    Sawa,Kiminari

    2012-08-01

    Full Text Available The relationship between pancreatic fibrosis and apoptosis of pancreatic acinar cells has not been fully elucidated. We reported that taurine had an anti-fibrotic effect in a dibutyltin dichloride (DBTC-chronic pancreatitis model. However, the effect of taurine on apoptosis of pancreatic acinar cells is still unclear. Therefore, we examined apoptosis in DBTC-chronic pancreatitis and in the AR42J pancreatic acinar cell line with/without taurine. Pancreatic fibrosis was induced by a single administration of DBTC. Rats were fed a taurine-containing diet or a normal diet and were sacrificed at day 5. The AR42J pancreatic acinar cell line was incubated with/without DBTC with taurine chloramines. Apoptosis was determined by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL assay. The expression of Bad and Bcl-2 proteins in the AR42J cells lysates was detected by Western blot analysis. The apoptotic index of pancreatic acinar cells in DBTC-administered rats was significantly increased. Taurine treatment inhibited pancreatic fibrosis and apoptosis of acinar cells induced by DBTC. The number of TUNEL-positive cells in the AR42J pancreatic acinar cell lines was significantly increased by the addition of DBTC. Incubation with taurine chloramines ameliorated these changes. In conclusion, taurine inhibits apoptosis of pancreatic acinar cells and pancreatitis in experimental chronic pancreatitis.

  20. Pancreatic acinar cells-derived cyclophilin A promotes pancreatic damage by activating NF-κB pathway in experimental pancreatitis

    International Nuclear Information System (INIS)

    Highlights: • CypA is upregulated in experimental pancreatitis. • CCK induces expression and release of CypA in acinar cell in vitro. • rCypA aggravates CCK-induced acinar cell death and inflammatory cytokine production. • rCypA activates the NF-κB pathway in acinar cells in vitro. - Abstract: Inflammation triggered by necrotic acinar cells contributes to the pathophysiology of acute pancreatitis (AP), but its precise mechanism remains unclear. Recent studies have shown that Cyclophilin A (CypA) released from necrotic cells is involved in the pathogenesis of several inflammatory diseases. We therefore investigated the role of CypA in experimental AP induced by administration of sodium taurocholate (STC). CypA was markedly upregulated and widely expressed in disrupted acinar cells, infiltrated inflammatory cells, and tubular complexes. In vitro, it was released from damaged acinar cells by cholecystokinin (CCK) induction. rCypA (recombinant CypA) aggravated CCK-induced acinar cell necrosis, promoted nuclear factor (NF)-κB p65 activation, and increased cytokine production. In conclusion, CypA promotes pancreatic damage by upregulating expression of inflammatory cytokines of acinar cells via the NF-κB pathway

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

    Science.gov (United States)

    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.

  2. A Patient of Pancreatic Acinar Cell Carcinoma with Dilated Esophagogastric Vessels

    Directory of Open Access Journals (Sweden)

    Masakuni Fujii

    2015-07-01

    Full Text Available Left portal hypertension and splenic vein occlusion commonly occur with pancreatic tumors, however these signs are rarely observed in patients with acinar cell carcinoma. This report describes a rare left portal hypertension in a patient who presented with a dilated esophagogastric vein upon esophagogastroduodenoscopic examination of a gastric polyp. A contrast-enhanced computed tomography scan revealed a pancreatic tumor, with obstruction of the splenic vein and portal-systemic shunt. The patient was diagnosed with an acinar cell carcinoma of the pancreatic tail. This patient highlights that pancreatic acinar cell carcinoma should be considered as a differential diagnosis in patients with a dilated esophagogastric vein and without signs of liver disease.

  3. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice.

    Science.gov (United States)

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-Ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  4. Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

    Science.gov (United States)

    Miyazaki, Satsuki; Tashiro, Fumi; Miyazaki, Jun-ichi

    2016-01-01

    A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes. PMID:27526291

  5. Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells

    Directory of Open Access Journals (Sweden)

    Maria del Rosario Espinoza-Mellado

    2013-12-01

    Full Text Available Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a “vacuolating factor” in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae .

  6. Acinar cell apoptosis in Serpini2-deficient mice models pancreatic insufficiency.

    Directory of Open Access Journals (Sweden)

    Stacie K Loftus

    2005-09-01

    Full Text Available Pancreatic insufficiency (PI when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman-Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq, exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome

  7. Acinar Cell Apoptosis in Serpini2-DeficientMice Models Pancreatic Insufficiency.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Pancreatic insufficiency (PI when left untreated results in a state of malnutrition due to an inability to absorb nutrients. Frequently, PI is diagnosed as part of a larger clinical presentation in cystic fibrosis or Shwachman-Diamond syndrome. In this study, a mouse model for isolated exocrine PI was identified in a mouse line generated by a transgene insertion. The trait is inherited in an autosomal recessive pattern, and homozygous animals are growth retarded, have abnormal immunity, and have reduced life span. Mice with the disease locus, named pequeño (pq, exhibit progressive apoptosis of pancreatic acinar cells with severe exocrine acinar cell loss by 8 wk of age, while the islets and ductal tissue persist. The mutation in pq/pq mice results from a random transgene insertion. Molecular characterization of the transgene insertion site by fluorescent in situ hybridization and genomic deletion mapping identified an approximately 210-kb deletion on Chromosome 3, deleting two genes. One of these genes, Serpini2, encodes a protein that is a member of the serpin family of protease inhibitors. Reintroduction of only the Serpini2 gene by bacterial artificial chromosome transgenic complementation corrected the acinar cell defect as well as body weight and immune phenotypes, showing that deletion of Serpini2 causes the pequeño phenotype. Dietary supplementation of pancreatic enzymes also corrected body size, body weight, and immunodeficiency, and increased the life span of Serpini2-deficient mice, despite continued acinar cell loss. To our knowledge, this study describes the first characterized genetic animal model for isolated PI. Genetic complementation of the transgene insertion mutant demonstrates that Serpini2 deficiency directly results in the acinar cell apoptosis, malabsorption, and malnutrition observed in pq/pq mice. The rescue of growth retardation, immunodeficiency, and mortality by either Serpini2 bacterial artificial chromosome

  8. Transdifferentiation of human amniotic epithelial cells into acinar cells using a double-chamber system.

    Science.gov (United States)

    Huang, Gui-Lin; Zhang, Ni-Ni; Wang, Jun-Sheng; Yao, Li; Zhao, Yu-Jie; Wang, Yu-Ying

    2012-08-01

    This study investigated the transdifferentiation of stem cells from human amnion tissue into functional acinar cells (ACs) using a co-culture system. Human amniotic epithelial cells (hAECs) were isolated from amnion tissue by mechanical mincing and enzymatic digestion. After primary culture, the phenotype of the cells was identified by flow cytometry (FCM) and immunocytochemical staining. hAECs were co-cultured with submandibular gland acinar cells of SD rats using a double-chamber system. The expression of α-amylase was determined by immunocytochemical method and fluorescent real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) after induction for 1 and 2 weeks, respectively. Digestion with trypsin is an effective method for isolating hAECs from amnion tissue. These cells were positive for CD29 and CK19 and weakly positive for CD44 and α-amylase. Within 2 weeks, α-amylase in hAECs increased with induction time. The expression of α-amylase in hAECs was increased 3.38-fold after co-culturing for 1 week. This ratio increased to 6.6-fold, and these cells were positive for mucins, after co-culturing for 2 weeks. hAECs possess the potential to differentiate into ACs in vitro. They might be a stem cell resource for clinical applications of cell replacement therapy in salivary gland dysfunction diseases. PMID:22800093

  9. Salivary gland acinar cells regenerate functional glandular structures in modified hydrogels

    Science.gov (United States)

    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

  10. Epiregulin is critical for the acinar cell regeneration of the submandibular gland in a mouse duct ligation model.

    Science.gov (United States)

    Nagai, Koichi; Arai, Hideo; Okudera, Michisato; Yamamura, Takashi; Oki, Hidero; Komiyama, Kazuo

    2014-05-01

    Acinar cell regeneration from tubular structures has been reported to occur in duct-deligated salivary glands. However, the detailed process of acinar cell regeneration has not been clarified. We have developed a mouse duct ligation model to clarify the mechanisms underlying acinar cell regeneration, and we analyzed the epidermal growth factor receptor (EGFR) and epidermal growth factor (EGF) ligands using the model. We studied these ligands expressions in the course of acinar cell regeneration using immunohistochemistry and RT-PCR methods. In the duct-ligated portion of the submandibular gland (SMG) that underwent atrophy, newly formed acinar cells were observed arising from the tubular structures after the release of the duct obstruction. The constitutive expression of EGFR was observed by immunohistochemistry in both the duct-ligated and duct-deligated animals as well as in normal controls. The EGFR phosphorylation detected on the tubular structures after duct ligation paralleled the acinar cell regeneration. RT-PCR showed an increase in the epiregulin and heparin-binding EGF levels from day 0 to day 3 after the release of the duct obstruction. The EGF level was increased only after day 7. In vitro, cultured cells isolated from ligated SMGs proliferated and produced EGF ligands following the addition of epiregulin to the culture medium. These findings suggest that the tubular structures localized in an atrophic gland are the source of acinar cell regeneration of the salivary gland. The induction of EGF ligands, in particular epiregulin, may play an important role in acinar cell regeneration in this model.

  11. Analysis of changes in the expression pattern of claudins using salivary acinar cells in primary culture.

    Science.gov (United States)

    Fujita-Yoshigaki, Junko

    2011-01-01

    Primary saliva is produced from blood plasma in the acini of salivary glands and is modified by ion adsorption and secretion as the saliva passes through the ducts. In rodents, acinar cells of salivary glands express claudin-3 but not claudin-4, whereas duct cells express both claudins-3 and -4. The distinct claudin expression patterns may reflect differences in the permeability of tight junctions between acinar and duct cells. To analyze the role of claudins in salivary glands, we established a system for the primary culture of parotid acinar cells, where the expression patterns of claudins are remarkably changed. Real-time RT-PCR and immunoblot analyses reveal that the expression levels of claudins-4 and -6 increased, whereas claudins-3 and -10 decreased. We found that the signal to induce those changes is triggered during cell isolation and is mediated by Src and p38 MAP kinase. Here, we introduce the methods used to determine the signal pathway that induces the change in claudin expression.

  12. Different cell death modes of pancreatic acinar cells on macrophage activation in rats

    Institute of Scientific and Technical Information of China (English)

    LIANG Tao; LIU Tie-fu; XUE Dong-bo; SUN Bei; SHI Li-jun

    2008-01-01

    Background The pathogenesis of acute pancreatitis is complex and largely unclear. The aim of this study was to explore the relationship between modes of cell death in pancreatic acinar cells, the release of cell contents and the inflammatory response of macrophagas.Methods Our experiment included four groups: group A (the control group), group B (AR42J cells overstimulated by caerulein), group C (AR42J cells treated with lipopolysaccharide and caerulein), and group D (AR42J cells treated with octreotide and caerulein). Apoptosis and oncosis, and the release of amylase and lactate dehydrogenase (LDH) from AR42J cells were detected. Rat macrophages were stimulated by 1 ml supematant of culture medium of AR42J cells.Finally, NF-кB activation and TNF-α and IL-1β secretion by macrophages were detected.Results Oncotlc cells in group C increased while apoptctic cells decreased (P <0.05); cells in group D had the inverse reaction. The release of amylase and LDH changed directly with the occurrence of oncosis. The transcription factor NF-кB was activated and secretion of TNF-α and IL-1β were significantly higher in group C than in group B (P <0.05); in group D, these actions were significantly lower than in group B (P<0.05). This trend was in line with changes in amylase and LDH production.Conclusion There is a close relationship between modes of pancreatic acinar cell death, the release of cell contents and the inflammatory reaction of macrophages.

  13. Modelling the transition from simple to complex Ca2+ oscillations in pancreatic acinar cells

    Indian Academy of Sciences (India)

    Neeraj Manhas; James Sneyd; K R Pardasani

    2014-06-01

    A mathematical model is proposed which systematically investigates complex calcium oscillations in pancreatic acinar cells. This model is based on calcium-induced calcium release via inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR) and includes calcium modulation of inositol (1,4,5) trisphosphate (IP3) levels through feedback regulation of degradation and production. In our model, the apical and the basal regions are separated by a region containing mitochondria, which is capable of restricting Ca2+ responses to the apical region. We were able to reproduce the observed oscillatory patterns, from baseline spikes to sinusoidal oscillations. The model predicts that calcium-dependent production and degradation of IP3 is a key mechanism for complex calcium oscillations in pancreatic acinar cells. A partial bifurcation analysis is performed which explores the dynamic behaviour of the model in both apical and basal regions.

  14. Regulating effects of arsenic trioxide on cell death pathways and inflammatory reactions of pancreatic acinar cells in rats

    Institute of Scientific and Technical Information of China (English)

    XUE Dong-bo; ZHANG Wei-hui; YUN Xiao-guang; SONG Chun; ZHENG Biao; SHI Xing-ye; WANG Hai-yang

    2007-01-01

    Background It is accepted that inflammatory cytokines play a key role in the development of acute pancreatitis, so blocking the initiation of inflammatory reactions may alleviate pathological changes of acute pancreatitis. We studied the regulatory effect of arsenic trioxide (As2O3) on apoptosis and oncosis of pancreatic acinar cells in vitro and in vivo and its therapeutic effect on acute pancreatitis.Methods Pancreatic acinar cells were isolated by collagenase digestion method. Apoptosis and oncosis of isolated pancreatic acinar cells were detected with Hoechst 33258+PI or Annexin V+PI double fluorescent staining. Amylase and lactate dehydrogenase release were measured. Acute pancreatitis was induced in Wistar rats by intraperitoneal injections of caerulein, and apoptosis was detected with terminal dUTP nick-end labeling method. Tumor necorsis factor α (TNF-α) mRNA, myeloperoxidase, nuclear factor-κB and histological grading of pancreatic damage were measured.Results There was an increased apoptosis but a decreased oncosis of pancreatic acinar cell after the treatment with As2O3. The levels of lactate dehydrogenase and amylase release were markedly decreased in As2O3 treated group.Myeloperoxidase content, TNF-α mRNA level, nuclear factor-κB activation and pathological score in As2O3 treated group were significantly lower than in the untreated group.Conclusions As2O3 can induce apoptosis and reduce oncosis of pancreatic acinar cell, thus resulting in reduced release of endocellular enzyme of acinar cells, reduced inflammatory cell infiltration and decreased the production of inflammatory cytokines, so that the outcome of alleviated pathological changes was finally achieved.

  15. Glycosylations in demilunar and central acinar cells of the submandibular salivary gland of ferret investigated by lectin histochemistry.

    Science.gov (United States)

    Triantafyllou, Asterios; Fletcher, David; Scott, John

    2004-09-01

    'Resting' submandibular salivary glands obtained post-mortem from mature ferrets of both sexes were examined here. The binding patterns of labelled lectins applied to paraffin sections of tissue slivers fixed in an aldehyde-HgCl2 mixture and the effects of pretreatment procedures on the results were assessed lightmicroscopically. Lectins with affinity for terminal GalNAc residues (DBA, SBA) bound preferentially to demilunar acinar cells which were also strongly reactive with Fuc-directed UEA I. In contrast, lectins with affinity for neuraminic acid (SNA, WGA) bound to central acinar cells where consistent binding of DBA and SNA occurred only after neuraminidase digestion, and variation in the binding of UEA I was seen. The reactivities corresponded with the distribution of secretory granules, but staining in Golgi-like areas occurred in central acinar cells with PNA lectin. The results suggest that glycosylations are more advanced in central than demilunar acinar cells of the ferret submandibular gland. Possibly demilunar and central acinar cells reflect phenotypic changes of a single secretory cell, the 'central' acinar phenotype being influenced by incorporation of neuraminic acid in glycoprotein side chains and by increased Golgi activity.

  16. Organelle selection determines agonist-specific Ca2+ signals in pancreatic acinar and beta cells

    OpenAIRE

    Yamasaki, M.; Masgrau, R.; Morgan, A. J.; Churchill, G. C.; Patel, S.; Ashcroft, S. J. H.; Galione, A

    2004-01-01

    How different extracellular stimuli can evoke different spatiotemporal Ca2+ signals is uncertain. We have elucidated a novel paradigm whereby different agonists use different Ca2+-storing organelles ("organelle seleetion") to evoke unique responses. Some agonists select the endoplasmic reticulum (ER), and others select lysosome-related (acidic) organelles, evoking spatial Ca2+ responses that mirror the organellar distribution. In pancreatic acinar cells, acetylcholine and bombesin exclusively...

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

    Science.gov (United States)

    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.

  18. Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo

    OpenAIRE

    Baumann, Bernd; Wagner, Martin; Aleksic, Tamara; von Wichert, Götz; Weber, Christoph K.; Adler, Guido; Wirth, Thomas

    2007-01-01

    Activation of the inhibitor of NF-κB kinase/NF-κB (IKK/NF-κB) system and expression of proinflammatory mediators are major events in acute pancreatitis. However, the in vivo consequences of IKK activation on the onset and progression of acute pancreatitis remain unclear. Therefore, we modulated IKK activity conditionally in pancreatic acinar cells. Transgenic mice expressing the reverse tetracycline-responsive transactivator (rtTA) gene under the control of the rat elastase promoter were gene...

  19. Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis.

    Science.gov (United States)

    Grabliauskaite, Kamile; Saponara, Enrica; Reding, Theresia; Bombardo, Marta; Seleznik, Gitta M; Malagola, Ermanno; Zabel, Anja; Faso, Carmen; Sonda, Sabrina; Graf, Rolf

    2016-02-01

    Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer.

  20. Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis.

    Science.gov (United States)

    Grabliauskaite, Kamile; Saponara, Enrica; Reding, Theresia; Bombardo, Marta; Seleznik, Gitta M; Malagola, Ermanno; Zabel, Anja; Faso, Carmen; Sonda, Sabrina; Graf, Rolf

    2016-02-01

    Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor-β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ-RII) using a Cre-LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein-induced pancreatitis. We show that TGFβ-RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ-RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell-autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ-RII signalling stimulates the expression of cyclin-dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ-RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ-based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer. PMID:26510396

  1. Role of endodermal cell vacuoles in shoot gravitropism.

    Science.gov (United States)

    Kato, Takehide; Morita, Miyo Terao; Tasaka, Masao

    2002-06-01

    In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.

  2. Cannabinoid receptors in submandibular acinar cells: functional coupling between saliva fluid and electrolytes secretion and Ca2+ signalling.

    Science.gov (United States)

    Kopach, Olga; Vats, Juliana; Netsyk, Olga; Voitenko, Nana; Irving, Andrew; Fedirko, Nataliya

    2012-04-15

    Cannabinoid receptors (CBRs) belong to the G protein-coupled receptor superfamily, and activation of CBRs in salivary cells inhibits agonist-stimulated salivation and modifies saliva content. However, the role of different CBR subtypes in acinar cell physiology and in intracellular signalling remains unclear. Here, we uncover functional CB(1)Rs and CB(2)Rs in acinar cells of rat submandibular gland and their essential role in saliva secretion. Pharmacological activation of CB(1)Rs and CB(2)Rs in the submandibular gland suppressed saliva outflow and modified saliva content produced by the submandibular gland in vivo. Using Na(+)-selective microelectrodes to record secretory Na(+) responses in the lumen of acini, we observed a reduction in Na(+) transport following the activation of CBRs, which was counteracted by the selective CB(1)R antagonist AM251. In addition, activation of CB(1)Rs or CB Rs caused inhibition of Na(+)-K(+) 2 -ATPase activity in microsomes derived from the gland tissue as well as in isolated acinar cells. Using a Ca(2+) imaging technique, we showed that activation of CB(1)Rs and CB(2)Rs alters [Ca(2+)](cyt) signalling in acinar cells by distinct pathways, involving Ca(2+) release from the endoplasmic reticulum (ER) and store-operated Ca(2+) entry (SOCE), respectively. Our data demonstrate the expression of CB(1)Rs and CB(2)Rs in acinar cells, and their involvement in the regulation of salivary gland functioning.

  3. Genetic deletion of Rab27B in pancreatic acinar cells affects granules size and has inhibitory effects on amylase secretion.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Lentz, Stephen I; Williams, John A

    2016-03-18

    Small G protein Rab27B is expressed in various secretory cell types and plays a role in mediating secretion. In pancreatic acinar cells, Rab27B was found to be expressed on the zymogen granule membrane and by overexpression to regulate the secretion of zymogen granules. However, the effect of Rab27B deletion on the physiology of pancreatic acinar cells is unknown. In the current study, we utilized the Rab27B KO mouse model to better understand the role of Rab27B in the secretion of pancreatic acinar cells. Our data show that Rab27B deficiency had no obvious effects on the expression of major digestive enzymes and other closely related proteins, e.g. similar small G proteins, such as Rab3D and Rab27A, and putative downstream effectors. The overall morphology of acinar cells was not changed in the knockout pancreas. However, the size of zymogen granules was decreased in KO acinar cells, suggesting a role of Rab27B in regulating the maturation of secretory granules. The secretion of digestive enzymes was moderately decreased in KO acini, compared with the WT control. These data indicate that Rab27B is involved at a different steps of zymogen granule maturation and secretion, which is distinct from that of Rab3D.

  4. 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.

    Science.gov (United States)

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

    2016-04-15

    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 [Ca(2+)] 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 Ca(2+)-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 Ca(2+) 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 Ca(2+)-dependent up-regulation of AQP5. These important findings reveal that the Ca(2+)-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.

  5. Antigen-presenting cells in parotid glands contain cystatin D originating from acinar cells.

    Science.gov (United States)

    Nashida, Tomoko; Sato, Ritsuko; Haga-Tsujimura, Maiko; Yoshie, Sumio; Yoshimura, Ken; Imai, Akane; Shimomura, Hiromi

    2013-02-01

    Cystatin D encoded by Cst5 is a salivary classified type II cystatin. We investigated the dynamism of cystatin D by examining the distribution of cystatin D protein and mRNA in rats, to identify novel functions. The simultaneous expression of Cst5 and cystatin D was observed in parotid glands, however in situ hybridization showed that only acinar cells produced cystatin D. Synthesized cystatin D was localized in small vesicles and secreted from the apical side to the saliva, and from the basolateral side to the extracellular region, a second secretory pathway for cystatin D. We also identified antigen-presenting cells in the parotid glands that contained cystatin D without the expression of Cst5, indicating the uptake of cystatin D from the extracellular region. Cystatin D was detected in blood serum and renal tubular cells with megalin, indicating the circulation of cystatin D through the body and uptake by renal tubular cells. Thus, the novel dynamism of cystatin D was shown and a function for cystatin D in the regulation of antigen-presenting cell activity was proposed.

  6. A tetanus toxin sensitive protein other than VAMP 2 is required for exocytosis in the pancreatic acinar cell.

    Science.gov (United States)

    Padfield, P J

    2000-11-01

    The neurotoxin sensitivity of regulated exocytosis in the pancreatic acinar cell was investigated using streptolysin-O permeabilized pancreatic acini. Treatment of permeabilized acini with botulinum toxin B (BoNT/B) or botulinum toxin D (BoNT/D) had no detectable effect on Ca(2+)-dependent amylase secretion but did result in the complete cleavage of VAMP 2. In comparison, tetanus toxin (TeTx) treatment both significantly inhibited Ca(2+)-dependent amylase secretion and cleaved VAMP 2. These results indicate that regulated exocytosis in the pancreatic acinar cell requires a tetanus toxin sensitive protein(s) other than VAMP 2.

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

    OpenAIRE

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

    2015-01-01

    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 ide...

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

    Science.gov (United States)

    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

    2016-07-01

    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

  9. β-catenin is selectively required for the expansion and regeneration of mature pancreatic acinar cells in mice

    Directory of Open Access Journals (Sweden)

    Matthew D. Keefe

    2012-07-01

    The size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt–β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt–β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.

  10. A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

    Directory of Open Access Journals (Sweden)

    Melissa A Metzler

    Full Text Available The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1 activates the Mist1 promoter [corrected]. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a

  11. The effect of irradiation on the intracellular transportation of the parotid gland acinar cells in the mouse. Localization of monosaccharides studied by electron microscopic autoradiography

    Energy Technology Data Exchange (ETDEWEB)

    Matsunaga, Hajime (Nippon Dental Univ., Tokyo (Japan))

    1994-06-01

    The present study was designed to investigate the effects of radiation on the ability to ingest monosaccharides and intracellular transportation in the parotid gland in mice. The submandibular regions, including the parotid gland, was exposed to 10 Gy of X-rays. Three days after irradiation, the localization of reducing silver grains in organelles was determined, using electron microscopic autoradiography with H-3 labeled galactosamine, glucosamine, fucose, and mannose. In the non-irradiated group, the proportion of reducing silver grains in the acinar cells began to increase 15 min after administration of monosaccharides, reached a peak at 180 min, and thereafter decreased. Similar findings were observed in the irradiated group, although the values were lower than the non-irradiated group. The proportion of reducing silver grains in the endoplasmic reticulum reached a peak at 15 min in both the non-irradiated and irradiated groups, and gradually decreased until 120 min. Thereafter, it became almost constant and low, but the proportion in the irradiated group was slightly higher than in the non-irradiated group. The proportion of reducing silver grains in the Golgi apparatus was maximum at 60 min in the non-irradiated group, and gradually decreased until 360 min. A similar tendency was seen in the irradiated group, although its variation was not so marked as in the non-irradiated group. The proportion of reducing silver grains in the condensing vacuoles was maximum at 120 min, and thereafter, it decreased; the decrease was only slight in the irradiated group. The proportion of reducing silver grains in secretory granules increased with time in both the non-irradiated and irradiated groups, although this was only slight in the irradiated group, and reached a peak at 360 min. Transportation of monosaccharides in an acinar cell was found to be delayed by irradiation. (N.K.).

  12. The effect of irradiation on the intracellular transportation of the parotid gland acinar cells in the mouse. Localization of monosaccharides studied by electron microscopic autoradiography

    International Nuclear Information System (INIS)

    The present study was designed to investigate the effects of radiation on the ability to ingest monosaccharides and intracellular transportation in the parotid gland in mice. The submandibular regions, including the parotid gland, was exposed to 10 Gy of X-rays. Three days after irradiation, the localization of reducing silver grains in organelles was determined, using electron microscopic autoradiography with H-3 labeled galactosamine, glucosamine, fucose, and mannose. In the non-irradiated group, the proportion of reducing silver grains in the acinar cells began to increase 15 min after administration of monosaccharides, reached a peak at 180 min, and thereafter decreased. Similar findings were observed in the irradiated group, although the values were lower than the non-irradiated group. The proportion of reducing silver grains in the endoplasmic reticulum reached a peak at 15 min in both the non-irradiated and irradiated groups, and gradually decreased until 120 min. Thereafter, it became almost constant and low, but the proportion in the irradiated group was slightly higher than in the non-irradiated group. The proportion of reducing silver grains in the Golgi apparatus was maximum at 60 min in the non-irradiated group, and gradually decreased until 360 min. A similar tendency was seen in the irradiated group, although its variation was not so marked as in the non-irradiated group. The proportion of reducing silver grains in the condensing vacuoles was maximum at 120 min, and thereafter, it decreased; the decrease was only slight in the irradiated group. The proportion of reducing silver grains in secretory granules increased with time in both the non-irradiated and irradiated groups, although this was only slight in the irradiated group, and reached a peak at 360 min. Transportation of monosaccharides in an acinar cell was found to be delayed by irradiation. (N.K.)

  13. Tmem16A encodes the Ca2+-activated Cl- channel in mouse submandibular salivary gland acinar cells.

    Science.gov (United States)

    Romanenko, Victor G; Catalán, Marcelo A; Brown, David A; Putzier, Ilva; Hartzell, H Criss; Marmorstein, Alan D; Gonzalez-Begne, Mireya; Rock, Jason R; Harfe, Brian D; Melvin, James E

    2010-04-23

    Activation of an apical Ca(2+)-dependent Cl(-) channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca(2+)-gated Cl(-) currents generated by Tmem16A and Best2, members from two distinct families of Ca(2+)-activated Cl(-) channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca(2+)-activated Cl(-) currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca(2+)-activated Cl(-) current found in native salivary acinar cells. Best2(-/-) and Tmem16A(-/-) mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca(2+)-activated Cl(-) current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A(-/-) mice lacked a Ca(2+)-activated Cl(-) current and a Ca(2+)-mobilizing agonist failed to stimulate Cl(-) efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2(-/-) mice. Our results demonstrate that both Tmem16A and Best2 generate Ca(2+)-activated Cl(-) current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca(2+)-activated Cl(-) channel complex that is essential for saliva production by the submandibular gland.

  14. Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells.

    Science.gov (United States)

    Rosado, Juan A; Redondo, Pedro C; Salido, Ginés M; Sage, Stewart O; Pariente, Jose A

    2005-01-01

    We recently reported that store-operated Ca(2+) entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca(2+) channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca(2+) entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca(2+) influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca(2+) store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

  15. Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

    Science.gov (United States)

    Nishiyama, Yuko; Ohmichi, Tomohiro; Kazami, Sayaka; Iwasaki, Hiroki; Mano, Kousuke; Nagumo, Yoko; Kudo, Fumitaka; Ichikawa, Sosaku; Iwabuchi, Yoshiharu; Kanoh, Naoki; Eguchi, Tadashi; Osada, Hiroyuki; Usui, Takeo

    2016-05-01

    Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity. PMID:27104762

  16. Variations in the expression and distribution pattern of AQP5 in acinar cells of patients with sialadenosis.

    Science.gov (United States)

    Teymoortash, Afshin; Wiegand, Susanne; Borkeloh, Martin; Bette, Michael; Ramaswamy, Annette; Steinbach-Hundt, Silke; Neff, Andreas; Werner, Jochen A; Mandic, Robert

    2012-01-01

    Previously, we pointed out on a possible role of aquaporin 5 (AQP5) in the development of sialadenosis. The goal of the present study was to further assess the association of AQP5 in the development of this salivary gland disease. The acinar diameter and mean surface area appeared elevated in sialadenosis tissues, which is a typical observation in this disease. AQP5 expression was evaluated by immunohistochemistry using tissue samples derived from salivary glands of patients with confirmed sialadenosis either as a primary diagnosis or as a secondary diagnosis within the framework of other salivary gland diseases. Normal salivary gland tissue served as a control. In sialadenosis tissues, the AQP5 signal at the apical plasma membrane of acinar cells frequently appeared stronger compared with that in normal salivary glands. In addition, the distribution of AQP5 at the apical region seemed to differ between normal and sialadenosis tissues, where AQP5 frequently was diffusely distributed near or at the apical plasma membrane of the acinar cells in contrast to normal controls where the AQP5 signal was strictly confined to the apical plasma membrane. These observations suggest that sialadenosis is associated with a different AQP5 expression and distribution pattern in salivary acinar cells.

  17. Long-term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands.

    Science.gov (United States)

    Bighetti, Bruna B; d Assis, Gerson F; Vieira, Danilo C; Violato, Natalia M; Cestari, Tania M; Taga, Rumio; Bosqueiro, José R; Rafacho, Alex

    2014-10-01

    Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long-term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P glands (P salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P acinar cells was increased in the submandibular glands of the DEX rats (P glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long-term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland-specific response to GCs. Our data emphasize that GC-based therapies and insulin-resistant states have a negative impact on salivary gland homeostasis.

  18. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca2+ oscillations in mouse pancreatic acinar cells

    Science.gov (United States)

    Huang, Zebing; Wang, Haiyan; Wang, Jingke; Zhao, Mengqin; Sun, Nana; Sun, Fangfang; Shen, Jianxin; Zhang, Haiying; Xia, Kunkun; Chen, Dejie; Gao, Ming; Hammer, Ronald P.; Liu, Qingrong; Xi, Zhengxiong; Fan, Xuegong; Wu, Jie

    2016-01-01

    Emerging evidence demonstrates that the blockade of intracellular Ca2+ signals may protect pancreatic acinar cells against Ca2+ overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca2+ signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca2+ oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca2+ oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca2+ oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca2+ oscillations and L-arginine-induced enhancement of Ca2+ signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis. PMID:27432473

  19. Cannabinoid receptor subtype 2 (CB2R) agonist, GW405833 reduces agonist-induced Ca(2+) oscillations in mouse pancreatic acinar cells.

    Science.gov (United States)

    Huang, Zebing; Wang, Haiyan; Wang, Jingke; Zhao, Mengqin; Sun, Nana; Sun, Fangfang; Shen, Jianxin; Zhang, Haiying; Xia, Kunkun; Chen, Dejie; Gao, Ming; Hammer, Ronald P; Liu, Qingrong; Xi, Zhengxiong; Fan, Xuegong; Wu, Jie

    2016-01-01

    Emerging evidence demonstrates that the blockade of intracellular Ca(2+) signals may protect pancreatic acinar cells against Ca(2+) overload, intracellular protease activation, and necrosis. The activation of cannabinoid receptor subtype 2 (CB2R) prevents acinar cell pathogenesis in animal models of acute pancreatitis. However, whether CB2Rs modulate intracellular Ca(2+) signals in pancreatic acinar cells is largely unknown. We evaluated the roles of CB2R agonist, GW405833 (GW) in agonist-induced Ca(2+) oscillations in pancreatic acinar cells using multiple experimental approaches with acute dissociated pancreatic acinar cells prepared from wild type, CB1R-knockout (KO), and CB2R-KO mice. Immunohistochemical labeling revealed that CB2R protein was expressed in mouse pancreatic acinar cells. Electrophysiological experiments showed that activation of CB2Rs by GW reduced acetylcholine (ACh)-, but not cholecystokinin (CCK)-induced Ca(2+) oscillations in a concentration-dependent manner; this inhibition was prevented by a selective CB2R antagonist, AM630, or was absent in CB2R-KO but not CB1R-KO mice. In addition, GW eliminated L-arginine-induced enhancement of Ca(2+) oscillations, pancreatic amylase, and pulmonary myeloperoxidase. Collectively, we provide novel evidence that activation of CB2Rs eliminates ACh-induced Ca(2+) oscillations and L-arginine-induced enhancement of Ca(2+) signaling in mouse pancreatic acinar cells, which suggests a potential cellular mechanism of CB2R-mediated protection in acute pancreatitis. PMID:27432473

  20. A resected case of symptomatic acinar cell cystadenoma of the pancreas displacing the main pancreatic duct.

    Science.gov (United States)

    Tanaka, Haruyoshi; Hatsuno, Tsuyoshi; Kinoshita, Mitsuru; Hasegawa, Kazuya; Ishihara, Hiromasa; Takano, Nao; Shimoyama, Satofumi; Nakayama, Hiroshi; Kataoka, Masato; Ichihara, Shu; Kanda, Mitsuro; Kodera, Yasuhiro; Kondo, Ken

    2016-12-01

    Acinar cell cystadenoma (ACA) of the pancreas has been newly recognized as an entity by the World Health Organization (WHO) definition (2010), and its pathogenesis has not been known adequately because of the rarity. Here, we report a case of a 22-year-old female who had been followed up for a cystic lesion at the tail of the pancreas pointed out by a screening computed tomography (CT) scan 7 years ago. The tumor grew in size from 3.3 to 5.1 cm in diameter for 6 years (0.3 cm per year). Particularly, it rapidly grew up to 6.3 cm in the latest 3 months in concurrence with the emergence of epigastralgia. A contrasted CT scan revealed the irregularly formed, multilocular cystic tumor having thin septum and calcification. The intratumoral magnetic resonance imaging intensity in the T1 and T2 weighted images were low and high, respectively. No communications between the tumor and the main pancreatic duct (MPD) were found, but the tumor displaced the MPD. She underwent surgical resection because the tumor was growing, turned symptomatic, and it seemed difficult to be diagnosed correctly until totally biopsied. Spleen-preserved distal pancreatectomy was performed. It was pathologically diagnosed as ACA; the cyst was lined by cells with normal acinar differentiation; cuboidal cells with round, basally oriented nuclei and eosinophilic granules in its apical cytoplasm. The abdominal pain has disappeared, and no recurrences have been found during a 5-year follow-up. Clinicians are recommended to consider an ACA as one of differential diagnoses of cystic tumors of the pancreas to provide appropriate diagnostics and therapeutics. PMID:27108123

  1. Ascl3 expression marks a progenitor population of both acinar and ductal cells in mouse salivary glands.

    Science.gov (United States)

    Bullard, Tara; Koek, Laurie; Roztocil, Elisa; Kingsley, Paul D; Mirels, Lily; Ovitt, Catherine E

    2008-08-01

    Ascl3, also know as Sgn1, is a member of the mammalian achaete scute (Mash) gene family of transcription factors, which have been implicated in cell fate specification and differentiation. In the mouse salivary gland, expression of Ascl3 is restricted to a subset of duct cells. Salivary gland function depends on the secretory acinar cells, which are responsible for saliva formation, and duct cells, which modify the saliva and conduct it to the oral cavity. The salivary gland ducts are also the putative site of progenitor cells in the adult gland. Using a Cre recombinase-mediated reporter system, we followed the fate of Ascl3-expressing cells after the introduction of an EGFP-Cre expression cassette into the Ascl3 locus by homologous recombination. Lineage tracing shows that these cells are progenitors of both acinar and ductal cell types in all three major salivary glands. In the differentiated progeny, expression of Ascl3 is down-regulated. These data directly demonstrate a progenitor-progeny relationship between duct cells and the acinar cell compartment, and identify a population of multipotent progenitor cells, marked by expression of Ascl3, which is capable of generating both gland cell types. We conclude that Ascl3-expressing cells contribute to the maintenance of the adult salivary glands.

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

    Directory of Open Access Journals (Sweden)

    Aljona Gaiko-Shcherbak

    Full Text Available 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.

  3. Uptake and metabolism of D-glucose in isolated acinar and ductal cells from rat submandibular glands.

    Science.gov (United States)

    Cetik, Sibel; Rzajeva, Aigun; Hupkens, Emeline; Malaisse, Willy J; Sener, Abdullah

    2014-07-01

    The present study deals with the possible effects of selected environmental agents upon the uptake and metabolism of d-glucose in isolated acinar and ductal cells from the rat submandibular salivary gland. In acinar cells, the uptake of d-[U-(14) C]glucose and its non-metabolised analogue 3-O-[(14) C-methyl]-d-glucose was not affected significantly by phloridzin (0.1 mM) or substitution of extracellular NaCl (115 mM) by an equimolar amount of CsCl, whilst cytochalasin B (20 μM) decreased significantly such an uptake. In ductal cells, both phloridzin and cytochalasin B decreased the uptake of d-glucose and 3-O-methyl-d-glucose. Although the intracellular space was comparable in acinar and ductal cells, the catabolism of d-glucose (2.8 or 8.3 mM) was two to four times higher in ductal cells than in acinar cells. Phloridzin (0.1 mM), ouabain (1.0 mM) and cytochalasin B (20 μM) all impaired d-glucose catabolism in ductal cells. Such was also the case in ductal cells incubated in the absence of extracellular Ca(2+) or in media in which NaCl was substituted by CsCl. It is proposed that the ductal cells in the rat submandibular gland are equipped with several systems mediating the insulin-sensitive, cytochalasin B-sensitive and phloridzin-sensitive transport of d-glucose across the plasma membrane.

  4. TNF-α inhibits aquaporin 5 expression in human salivary gland acinar cells via suppression of histone H4 acetylation.

    Science.gov (United States)

    Yamamura, Yoshiko; Motegi, Katsumi; Kani, Kouichi; Takano, Hideyuki; Momota, Yukihiro; Aota, Keiko; Yamanoi, Tomoko; Azuma, Masayuki

    2012-08-01

    Sjögren's syndrome is a systemic autoimmune disease characterized by reductions in salivary and lacrimal secretions. The mechanisms underlying these reductions remain unclear. We have previously shown that TNF-α plays an important role in the destruction of acinar structures. Here we examined TNF-α's function in the expression of aquaporin (AQP) 5 in human salivary gland acinar cells. Immortalized human salivary gland acinar (NS-SV-AC) cells were treated with TNF-α, and then the expression levels of AQP5 mRNA and protein were analysed. In addition, the mechanisms underlying the reduction of AQP5 expression by TNF-α treatment were investigated. TNF-α-treatment of NS-SV-AC cells significantly suppressed the expression levels of AQP5 mRNA and protein, and reduced the net fluid secretion rate. We examined the expression and activation levels of DNA methyltransferases (Dnmts) in NS-SV-AC cells treated with TNF-α. However, no significant changes were observed in the expression or activation levels of Dnmt1, Dnmt3a or Dnmt3b. Although we also investigated the role of NF-κB activity in the TNF-α-induced suppression of AQP5 expression in NS-SV-AC cells, we detected similar TNF-α suppression of AQP5 expression in non-transfected cells and in a super-repressor form of IκBα cDNA-transfected cell clones. However, interestingly, chromatin immunoprecipitation analysis demonstrated a remarkable decrease in levels of acetylated histone H4 associated with the AQP5 gene promoter after treatment with TNF-α in NS-SV-AC cells. Therefore, our results may indicate that TNF-α inhibition of AQP5 expression in human salivary gland acinar cells is due to the epigenetic mechanism by suppression of acetylation of histone H4.

  5. Increase in muscarinic stimulation-induced Ca(2+) response by adenovirus-mediated Stim1-mKO1 gene transfer to rat submandibular acinar cells in vivo.

    Science.gov (United States)

    Morita, Takao; Nezu, Akihiro; Tojyo, Yosuke; Tanimura, Akihiko

    2013-10-01

    Adenoviruses have been used for gene transfer to salivary gland cells in vivo. Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca(2+) responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca(2+) response was notable upon weak muscarinic stimulation and was attributed to increased Ca(2+) release from internal stores and increased Ca(2+) entry. The basal Ca(2+) level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca(2+) concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP3) produced similar effects on the release of Ca(2+) from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo, and may be applied to increase salivary secretions in experimental animals and human in future.

  6. Rab27A Is Present in Mouse Pancreatic Acinar Cells and Is Required for Digestive Enzyme Secretion.

    Directory of Open Access Journals (Sweden)

    Yanan Hou

    Full Text Available The small G-protein Rab27A has been shown to regulate the intracellular trafficking of secretory granules in various cell types. However, the presence, subcellular localization and functional impact of Rab27A on digestive enzyme secretion by mouse pancreatic acinar cells are poorly understood. Ashen mice, which lack the expression of Rab27A due to a spontaneous mutation, were used to investigate the function of Rab27A in pancreatic acinar cells. Isolated pancreatic acini were prepared from wild-type or ashen mouse pancreas by collagenase digestion, and CCK- or carbachol-induced amylase secretion was measured. Secretion occurring through the major-regulated secretory pathway, which is characterized by zymogen granules secretion, was visualized by Dextran-Texas Red labeling of exocytotic granules. The minor-regulated secretory pathway, which operates through the endosomal/lysosomal pathway, was characterized by luminal cell surface labeling of lysosomal associated membrane protein 1 (LAMP1. Compared to wild-type, expression of Rab27B was slightly increased in ashen mouse acini, while Rab3D and digestive enzymes (amylase, lipase, chymotrypsin and elastase were not affected. Localization of Rab27B, Rab3D and amylase by immunofluorescence was similar in both wild-type and ashen acinar cells. The GTP-bound states of Rab27B and Rab3D in wild-type and ashen mouse acini also remained similar in amount. In contrast, acini from ashen mice showed decreased amylase release induced by CCK- or carbachol. Rab27A deficiency reduced the apical cell surface labeling of LAMP1, but did not affect that of Dextran-Texas Red incorporation into the fusion pockets at luminal surface. These results show that Rab27A is present in mouse pancreatic acinar cells and mainly regulates secretion through the minor-regulated pathway.

  7. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells.

    Science.gov (United States)

    Bhopale, Kamlesh K; Falzon, Miriam; Ansari, G A S; Kaphalia, Bhupendra S

    2014-04-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

  8. Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells

    Science.gov (United States)

    Bhopale, Kamlesh K.; Falzon, Miriam; Ansari, G. A. S.

    2016-01-01

    Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with l,10-PT + ethanol and ~1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I—III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol. PMID:24281792

  9. [Thapsigargin-sensitive and insensitive intracellular calcium stores in acinar cells of the submandibular salivary gland in rats].

    Science.gov (United States)

    Kopach, O V; Kruhlykov, I A; Voĭtenko, N V; Fedirko, N V

    2005-01-01

    Acinar cells of rat submandibular salivary gland are characterized by heterogeneity of intracellular Ca2+ stores. In the present work we have studied this heterogeneity using Arsenazo III dye to measure a cellular total calcium content and Fura-2/AM, to determine free cytosolic calcium concentration ([Ca2+]i). We have found that the amount of Ca2+ released by inhibition of Ca2+ ATPase of the ER with thapsigargin comprises approximately 30% of total ER calcium. This result was obtained in experiments on both intact and permeabilized acinar cells. We have also shown that both Ca2+ ATPase inhibition with thapsigargin and emptying the stores with acetylcholine (ACh) led to activation of store-operated Ca2+ influx (an increase in total calcium content of approximately 14%). In permeabilized cells application of ACh after preincubation with thapsigargin led to a further decrease in total cellular calcium content (approximately 38%). At the same time in intact cells it resulted in generation of [Ca2+]i transients with gradually decreasing amplitudes. Thus, ACh is capable of producing an additional release of Ca2+ from thapsigargin-insensitive stores. This additional release is IP3-dependent since it was completely blocked by heparin. We conclude that in acinar cells of rat submandibular gland thapsigargin-sensitive and thapsigargin-insensitive Ca2+ stores could exist.

  10. Mutant KRas-Induced Mitochondrial Oxidative Stress in Acinar Cells Upregulates EGFR Signaling to Drive Formation of Pancreatic Precancerous Lesions

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    Geou-Yarh Liou

    2016-03-01

    Full Text Available The development of pancreatic cancer requires the acquisition of oncogenic KRas mutations and upregulation of growth factor signaling, but the relationship between these is not well established. Here, we show that mutant KRas alters mitochondrial metabolism in pancreatic acinar cells, resulting in increased generation of mitochondrial reactive oxygen species (mROS. Mitochondrial ROS then drives the dedifferentiation of acinar cells to a duct-like progenitor phenotype and progression to PanIN. This is mediated via the ROS-receptive kinase protein kinase D1 and the transcription factors NF-κB1 and NF-κB2, which upregulate expression of the epidermal growth factor, its ligands, and their sheddase ADAM17. In vivo, interception of KRas-mediated generation of mROS reduced the formation of pre-neoplastic lesions. Hence, our data provide insight into how oncogenic KRas interacts with growth factor signaling to induce the formation of pancreatic cancer.

  11. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago

    NARCIS (Netherlands)

    Gavrin, A.Y.; Kaiser, B.N.; Geiger, D.; Tyerman, S.D.; Wen, Z.; Bisseling, T.; Fedorova, E.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 cell

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

    Science.gov (United States)

    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.

  13. Ca²⁺ signaling and regulation of fluid secretion in salivary gland acinar cells.

    Science.gov (United States)

    Ambudkar, Indu S

    2014-06-01

    Neurotransmitter stimulation of plasma membrane receptors stimulates salivary gland fluid secretion via a complex process that is determined by coordinated temporal and spatial regulation of several Ca(2+) signaling processes as well as ion flux systems. Studies over the past four decades have demonstrated that Ca(2+) is a critical factor in the control of salivary gland function. Importantly, critical components of this process have now been identified, including plasma membrane receptors, calcium channels, and regulatory proteins. The key event in activation of fluid secretion is an increase in intracellular [Ca(2+)] ([Ca(2+)]i) triggered by IP3-induced release of Ca(2+) from ER via the IP3R. This increase regulates the ion fluxes required to drive vectorial fluid secretion. IP3Rs determine the site of initiation and the pattern of [Ca(2+)]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+)]i and fluid secretion. This Ca(2+) influx pathway, store-operated calcium influx pathway (SOCE), has been studied in great detail and the regulatory mechanisms as well as key molecular components have now been identified. Orai1, TRPC1, and STIM1 are critical components of SOCE and among these, Ca(2+) entry via TRPC1 is a major determinant of fluid secretion. The receptor-evoked Ca(2+) signal in salivary gland acinar cells is unique in that it starts at the apical pole and then rapidly increases across the cell. The basis for the polarized Ca(2+) signal can be ascribed to the polarized arrangement of the Ca(2+) channels, transporters, and signaling proteins. Distinct localization of these proteins in the cell suggests compartmentalization of Ca(2+) signals during regulation of fluid secretion. This chapter will discuss new concepts and findings regarding the polarization and control of Ca(2+) signals in the regulation of fluid secretion.

  14. Apical Ca2+-activated potassium channels in mouse parotid acinar cells.

    Science.gov (United States)

    Almassy, Janos; Won, Jong Hak; Begenisich, Ted B; Yule, David I

    2012-02-01

    Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

  15. Atp2c2 Is Transcribed From a Unique Transcriptional Start Site in Mouse Pancreatic Acinar Cells.

    Science.gov (United States)

    Fenech, Melissa A; Sullivan, Caitlin M; Ferreira, Lucimar T; Mehmood, Rashid; MacDonald, William A; Stathopulos, Peter B; Pin, Christopher L

    2016-12-01

    Proper regulation of cytosolic Ca(2+) is critical for pancreatic acinar cell function. Disruptions in normal Ca(2+) concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca(2+) homeostasis by promoting rapid Ca(2+) movement. Determining how expression of Ca(2+) modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca(2+) ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2's 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca(2+) . RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca(2+) levels. Combined, these results suggest SPCA2C affects Ca(2+) homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca(2+) ATPases. J. Cell. Physiol. 231: 2768-2778, 2016. © 2016 Wiley Periodicals, Inc. PMID:27017909

  16. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

    Directory of Open Access Journals (Sweden)

    Tomasz J. Wodzicki

    2015-05-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

  17. Directed pancreatic acinar differentiation of mouse embryonic stem cells via embryonic signalling molecules and exocrine transcription factors.

    Directory of Open Access Journals (Sweden)

    Fabien Delaspre

    Full Text Available Pluripotent embryonic stem cells (ESC are a promising cellular system for generating an unlimited source of tissue for the treatment of chronic diseases and valuable in vitro differentiation models for drug testing. Our aim was to direct differentiation of mouse ESC into pancreatic acinar cells, which play key roles in pancreatitis and pancreatic cancer. To that end, ESC were first differentiated as embryoid bodies and sequentially incubated with activin A, inhibitors of Sonic hedgehog (Shh and bone morphogenetic protein (BMP pathways, fibroblast growth factors (FGF and retinoic acid (RA in order to achieve a stepwise increase in the expression of mRNA transcripts encoding for endodermal and pancreatic progenitor markers. Subsequent plating in Matrigel® and concomitant modulation of FGF, glucocorticoid, and folllistatin signalling pathways involved in exocrine differentiation resulted in a significant increase of mRNAs encoding secretory enzymes and in the number of cells co-expressing their protein products. Also, pancreatic endocrine marker expression was down-regulated and accompanied by a significant reduction in the number of hormone-expressing cells with a limited presence of hepatic marker expressing-cells. These findings suggest a selective activation of the acinar differentiation program. The newly differentiated cells were able to release α-amylase and this feature was greatly improved by lentiviral-mediated expression of Rbpjl and Ptf1a, two transcription factors involved in the maximal production of digestive enzymes. This study provides a novel method to produce functional pancreatic exocrine cells from ESC.

  18. Gramicidin-perforated Patch Recording Revealed the Oscillatory Nature of Secretory Cl− Movements in Salivary Acinar Cells

    OpenAIRE

    Sugita, Makoto; Hirono, Chikara; Shiba, Yoshiki

    2004-01-01

    Elevations of cytoplasmic free calcium concentrations ([Ca2+]i) evoked by cholinergic agonists stimulate isotonic fluid secretion in salivary acinar cells. This process is driven by the apical exit of Cl− through Ca2+-activated Cl− channels, while Cl− enters the cytoplasm against its electrochemical gradient via a loop diuretic-sensitive Na+-K+-2Cl− cotransporter (NKCC) and/or parallel operations of Cl−-HCO3 − and Na+-H+ exchangers, located in the basolateral membrane. To characterize the con...

  19. Differences in claudin synthesis in primary cultures of acinar cells from rat salivary gland are correlated with the specific three-dimensional organization of the cells.

    Science.gov (United States)

    Qi, Bing; Fujita-Yoshigaki, Junko; Michikawa, Hiromi; Satoh, Keitaro; Katsumata, Osamu; Sugiya, Hiroshi

    2007-07-01

    Tight junctions are essential for the maintenance of epithelial cell polarity. We have previously established a system for the primary culture of salivary parotid acinar cells that retain their ability to generate new secretory granules and to secrete proteins in a signal-dependent manner. Because cell polarity and cell-cell adhesion are prerequisites for the formation of epithelial tissues, we have investigated the structure of the tight junctions in these cultures. We have found two types of cellular organization in the culture: monolayers and semi-spherical clusters. Electron microscopy has revealed tight junctions near the apical region of the lateral membranes between cells in the monolayers and cells at the surface of the clusters. The cells in the interior of the clusters also have tight junctions and are organized around a central lumen. These interior cells retain more secretory granules than the surface or monolayer cells, suggesting that they maintain their original character as acinar cells. The synthesis of claudin-4 increases during culture, although it is not detectable in the cells immediately after isolation from the glands. Immunofluorescence microscopy has shown that claudin-4 is synthesized in the monolayers and at the surface of the clusters, but not inside the clusters. Only claudin-3, which is present in the original acinar cells following isolation and in the intact gland, has been detected inside the clusters. These results suggest that differences in claudin expression are related to the three-dimensional structures of the cell cultures and reflect their ability to function as acinar cells.

  20. Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns.

    Science.gov (United States)

    Chan, Yen-Hui; Huang, Tsung-Wei; Young, Tai-Horng; Lou, Pei-Jen

    2011-11-01

    Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.

  1. Vacuolization and apoptosis induced by nano-selenium in HeLa cell line

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Selenium(Se),a potential drug candidate for cancer prevention,has a special property:Its nutritional dosage and tolerable upper intake level appear in a narrow range,while the therapeutic use of this mineral may depend on a higher body intake level.Nano-selenium(nano-Se) particles,however,preserve the selenium element’s low toxicity characteristic but give a high biochemical activity effect of selenium compounds.In the present study different morphologies of synthesized nano-Se were evaluated concerning its anti-proliferation and apoptosis-inducing effect.Then nano-Se(sphere) were picked out to investigate its influence on two significant events involved in apoptosis,cell cycle arrest and mitochondrial membrane potential disruption.Furthermore,massive vacuolization of HeLa cells treated by nano-Se(sphere) was observed and more methods were used to measure the level of vacuolization.Such vacuolization needs energy supply and has been demonstrated to be related to Se endocytosis.These results suggest a possible mechanism to trigger apoptosis initiation.

  2. Postnatal Pancreas of Mice Contains Tripotent Progenitors Capable of Giving Rise to Duct, Acinar, and Endocrine Cells In Vitro.

    Science.gov (United States)

    Ghazalli, Nadiah; Mahdavi, Alborz; Feng, Tao; Jin, Liang; Kozlowski, Mark T; Hsu, Jasper; Riggs, Arthur D; Tirrell, David A; Ku, H Teresa

    2015-09-01

    Postnatal pancreas is a potential source for progenitor cells to generate endocrine β-cells for treating type 1 diabetes. However, it remains unclear whether young (1-week-old) pancreas harbors multipotent progenitors capable of differentiating into duct, acinar, and endocrine cells. Laminin is an extracellular matrix (ECM) protein important for β-cells' survival and function. We established an artificial extracellular matrix (aECM) protein that contains the functional IKVAV (Ile-Lys-Val-Ala-Val) sequence derived from laminin (designated aECM-lam). Whether IKVAV is necessary for endocrine differentiation in vitro is unknown. To answer these questions, we cultured single cells from 1-week-old pancreas in semi-solid media supplemented with aECM-lam, aECM-scr (which contains a scrambled sequence instead of IKVAV), or Matrigel. We found that colonies were generated in all materials. Individual colonies were examined by microfluidic reverse transcription-polymerase chain reaction, immunostaining, and electron microscopy analyses. The majority of the colonies expressed markers for endocrine, acinar, and ductal lineages, demonstrating tri-lineage potential of individual colony-forming progenitors. Colonies grown in aECM-lam expressed higher levels of endocrine markers Insulin1, Insulin2, and Glucagon compared with those grown in aECM-scr and Matrigel, indicating that the IKVAV sequence enhances endocrine differentiation. In contrast, Matrigel was inhibitory for endocrine gene expression. Colonies grown in aECM-lam displayed the hallmarks of functional β-cells: mature insulin granules and glucose-stimulated insulin secretion. Colony-forming progenitors were enriched in the CD133(high) fraction and among 230 micro-manipulated single CD133(high) cells, four gave rise to colonies that expressed tri-lineage markers. We conclude that young postnatal pancreas contains multipotent progenitor cells and that aECM-lam promotes differentiation of β-like cells in vitro. PMID

  3. Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2016-02-01

    Full Text Available The glutathione of the red beetroot vacuoles (Beta vulgaris L. was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT; the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB; the high-performance liquid chromatography (HPLC. The content of reduced (GSH and oxidized glutathione (GSSG differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.

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

    Science.gov (United States)

    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.

  5. Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: Procainamide-induced autophagic cell vacuolization

    International Nuclear Information System (INIS)

    Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent KM 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥ 24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking

  6. Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells.

    Science.gov (United States)

    Satoh, Keitaro; Seo, Yoshiteru; Matsuo, Shinsuke; Karabasil, Mileva Ratko; Matsuki-Fukushima, Miwako; Nakahari, Takashi; Hosoi, Kazuo

    2012-10-01

    In order to assess the contribution of the water channel aquaporin-5 (AQP5) to water transport by salivary gland acinar cells, we measured the cell volume and activation energy (E (a)) of diffusive water permeability in isolated parotid acinar cells obtained from AQP5-G103D mutant and their wild-type rats. Immunohistochemistry showed that there was no change induced by carbamylcholine (CCh; 1 μM) in the AQP5 detected in the acinar cells in the wild-type rat. Acinar cells from mutant rats, producing low levels of AQP5 in the apical membrane, showed a minimal increase in the AQP5 due to the CCh. In the wild-type rat, CCh caused a transient swelling of the acinus, followed by a rapid agonist-induced cell shrinkage, reaching a plateau at 30 s. In the mutant rat, the acinus did not swell by CCh challenge, and the agonist-induced cell shrinkage was delayed by 8 s, reaching a transient minimum at around 1 min, and recovered spontaneously even though CCh was persistently present. In the unstimulated wild-type acinar cells, E (a) was 3.4 ± 0.6 kcal mol(-1) and showed no detectable change after CCh stimulation. In the unstimulated mutant acinar cells, high E (a) value (5.9 ± 0.1 kcal mol(-1)) was detected and showed a minimal decrease after CCh stimulation (5.0 ± 0.3 kcal mol(-1)). These results suggested that AQP5 was the main pathway for water transport in the acinar cells and that it was responsible for the rapid agonist-induced acinar cell shrinkage and also necessary to keep the acinar cell volume reduced during the steady secretion in the wild-type rat.

  7. Ae4 (Slc4a9) Anion Exchanger Drives Cl- Uptake-dependent Fluid Secretion by Mouse Submandibular Gland Acinar Cells.

    Science.gov (United States)

    Peña-Münzenmayer, Gaspar; Catalán, Marcelo A; Kondo, Yusuke; Jaramillo, Yasna; Liu, Frances; Shull, Gary E; Melvin, James E

    2015-04-24

    Transcellular Cl(-) movement across acinar cells is the rate-limiting step for salivary gland fluid secretion. Basolateral Nkcc1 Na(+)-K(+)-2Cl(-) cotransporters play a critical role in fluid secretion by promoting the intracellular accumulation of Cl(-) above its equilibrium potential. However, salivation is only partially abolished in the absence of Nkcc1 cotransporter activity, suggesting that another Cl(-) uptake pathway concentrates Cl(-) ions in acinar cells. To identify alternative molecular mechanisms, we studied mice lacking Ae2 and Ae4 Cl(-)/HCO3 (-) exchangers. We found that salivation stimulated by muscarinic and β-adrenergic receptor agonists was normal in the submandibular glands of Ae2(-/-) mice. In contrast, saliva secretion was reduced by 35% in Ae4(-/-) mice. The decrease in salivation was not related to loss of Na(+)-K(+)-2Cl(-) cotransporter or Na(+)/H(+) exchanger activity in Ae4(-/-) mice but correlated with reduced Cl(-) uptake during β-adrenergic receptor activation of cAMP signaling. Direct measurements of Cl(-)/HCO3 (-) exchanger activity revealed that HCO3 (-)-dependent Cl(-) uptake was reduced in the acinar cells of Ae2(-/-) and Ae4(-/-) mice. Moreover, Cl(-)/HCO3 (-) exchanger activity was nearly abolished in double Ae4/Ae2 knock-out mice, suggesting that most of the Cl(-)/HCO3 (-) exchanger activity in submandibular acinar cells depends on Ae2 and Ae4 expression. In conclusion, both Ae2 and Ae4 anion exchangers are functionally expressed in submandibular acinar cells; however, only Ae4 expression appears to be important for cAMP-dependent regulation of fluid secretion.

  8. Involvement of M3 Cholinergic Receptor Signal Transduction Pathway in Regulation of the Expression of Chemokine MOB-1, MCP-1 Genes in Pancreatic Acinar Cells

    Institute of Scientific and Technical Information of China (English)

    郑海; 陈道达; 张景輝; 田原

    2004-01-01

    Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-κB and the expression of chemokine MOB-1, MCP-1genes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-κB was monitored by using electrophoretic mobility shift assay.The results showed that as compared with control group, M3 cholinergic receptor agonist (103mol/L, 104-4ol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10 -3mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-κB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10-3 mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10-5 mol/L atropine) or NF-κB inhibitor (10-2 mol/L PDTC) could obviously inhibit the activation of NF-κB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P <0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-1genes in pancreatic acinar cells in vitro through the activation of NF-κB.

  9. Formation of post-confluence structure in human parotid gland acinar cells on PLGA through regulation of E-cadherin.

    Science.gov (United States)

    Chan, Yen-Hui; Huang, Tsung-Wei; Chou, Ya-Shuan; Hsu, Sheng-Hao; Su, Wei-Fang; Lou, Pei-Jen; Young, Tai-Horng

    2012-01-01

    As a potential solution for patients to retrieve their lost salivary gland functions, tissue engineering of an auto-secretory device is profoundly needed. Under serum-free environment, primary human parotid gland acinar (PGAC) cells can be obtained. After reaching confluence, PGAC cells spontaneously form three-dimension (3D) cell aggregations, termed post-confluence structure (PCS), and change their behaviors. Poly (lactic-co-glycolic acid) (PLGA) has been widely used in the field of biomedical applications because of its biodegradable properties for desired functions. Nonetheless, the role of PLGA in facilitating PGAC cells to form PCS has seldom been explored to recover epithelial characteristics. In this study, PGAC cells were found to have a greater tendency to form PCS on PLGA than on tissue culture polystyrene (TCPS). By tracing cell migration paths and modulating E-cadherin activity with specific inhibitor or antibody, we demonstrated that the static force of homophilic interaction on surfaces of individual cells, but not the dynamics of cell migration, played a more important role in PCS formation. Thus, PLGA was successfully confirmed to support PGAC cells to form more PCS through the effects on enhancing E-cadherin expression, which is associated with FAK/ILK/Snail expression in PGAC cells. This result indicates that selective appropriate biomaterials may be potentially useful in generating 3D PCS on two-dimension (2D) substrate without fabricating a complex 3D scaffold.

  10. Effects of Baicalin on inflammatory mediators and pancreatic acinar cell apoptosis in rats with sever acute pancreatitis

    Directory of Open Access Journals (Sweden)

    zhang xiping

    2009-02-01

    Full Text Available

    • BACKGROUND: To investigate the effects of Baicalin and Octreotide on inflammatory mediators and pancreatic acinar cells apoptosis of rats with severe acute pancreatitis (SAP.
    • METHODS: SD rats were randomly divided into sham operated group (I group, model control group (II group, Baicalin treated group (III group and Octreotide treated group (IV group. Each group was also divided into subgroup of 3, 6 and 12 h (n = 15. The mortality rate, ascites/body weight ratio as well as the level of endotoxin, NO and ET-1 in blood were measured. The pathological severity score of pancreas, apoptotic indexes, and expression levels of Bax and Bcl-2 proteins in each group were investigated.
    • RESULTS: The survival rate of III and IV group has a significant difference compared with II group (P12 h < 0.05. The ascites volume, contents of inflammatory mediators in blood and pathological severity score of pancreas of III and IV group declined at different degrees compared to II group (P < 0.05, P < 0.01 or P < 0.001. Apoptotic index in III group was significantly higher than that in II group at 3 and 6 h (P3, 6 h < 0.05. Apoptotic index in IV group was significantly higher than that in II group at pancreatic tail at 6 h (P6 h < 0.05. Expression level of Bax in III group was significantly higher than that in II group (pancreatic head P3 h,6 h < 0.01, pancreatic tail P3 h < 0.001.
    • CONCLUSIONS: Compared with Octreotide in the treatment of SAP, the protective mechanisms of Baicalin include reducing the excessive inflammatory mediators’ release, inducing the pancreatic acinar cells apoptosis.
    • KEY WORDS: Severe acute pancreatitis, baicalin, octreotide, inflammatory mediators, apoptosis, tissue microarrays.

  11. A comparison study of pancreatic acinar cell carcinoma with ductal adenocarcinoma using computed tomography in Chinese patients

    Directory of Open Access Journals (Sweden)

    Wang Q

    2016-09-01

    Full Text Available Qingbing Wang,1,2 Xiaolin Wang,1,2 Rongfang Guo,2,3 Guoping Li1,2 1Department of Interventional Radiology, Zhongshan Hospital, Fudan University, 2Shanghai Institute of Medical Imaging, 3Department of Radiology, Zhongshan Hospital, Fudan University, Shanghai, People’s Republic of China Abstract: Pancreatic acinar cell carcinoma (ACC is a rare tumor that is difficult to diagnose preoperatively. The aim of this study was to evaluate and describe the computed tomography (CT features of ACC and compare the results with pancreatic ductal adenocarcinoma (DAC for improving preoperative diagnosis. The control group consisted of 34 patients with DAC collected from the pathology electronic database. The CT imaging from nine patients with pathologically confirmed ACC was retrospectively reviewed. Two radiologists independently assessed the tumor location, size, texture, and enhancement patterns. We found that 64.3% (9/14 of ACC tumors were homogeneous and 35.7% (5/14 had necrosis. The percentage of common bile duct and pancreatic ductal dilation was 14.3% (2/14 and 7.1% (1/14, respectively. The mean size of ACC was 50.1±24.2 mm. The mean attenuation of ACC was 35.4±3.9 Hounsfield unit (HU before enhancement, 73.1±42.9 HU in arterial phase, and 71.8±15.6 HU in port venous phase. It is difficult to distinguish ACC from DAC preoperatively only based on CT findings. However, compared with DAC, we found that ACC tumors are likely to be larger and contain more heterogeneous intratumoral necrotic hypovascular regions, and less pancreatic ductal and common biliary dilation. Keywords: acinar cell carcinoma, computed tomography, pancreatic ductal carcinoma, pancreas

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

    Science.gov (United States)

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    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

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

    Science.gov (United States)

    Teos, L Y; Zheng, C-Y; Liu, X; Swaim, W D; Goldsmith, C M; Cotrim, A P; Baum, B J; Ambudkar, I S

    2016-07-01

    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

  14. Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

    Institute of Scientific and Technical Information of China (English)

    Yumi Hirakawa; Toshihisa Nomura; Seiichiro Hasezawa; Takumi Higaki

    2015-01-01

    Vacuoles are suggested to play crucial roles in plant defense-related cel death. During programmed cel death, previous live cel imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole’s rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cel cycle-synchronized tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yel ow 2) cel s during cel death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90%of the cel s by 24 h. Prior to cel death, vacuole shape simplified and endoplasmic actin filaments disassembled;however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cel death.

  15. Protein kinase C expression in salivary gland acinar epithelial cells in non-obese diabetic mice, an experimental model for Sjögren's syndrome.

    Science.gov (United States)

    Tensing, E-K; Ma, J; Hukkanen, M; Fox, H S; Li, T-F; Törnwall, J; Konttinen, Y T

    2005-01-01

    We planned to investigate the expression of protein kinase C (PKC) isoforms in acinar epithelial cells of salivary glands in the non-obese diabetic (NOD) mouse to find out if they develop changes of the PKC system like those seen in the human counterpart, i.e. in Sjögren's syndrome. Parotid, submandibular, and sublingual glands from NOD and control BALB/c mice were stained with a panel of monoclonal antibodies directed against conventional (alpha, beta, and gamma), novel (delta, epsilon, and theta), and atypical (lambda and iota) PKC isoforms using the streptavidin/HRP method. Similarly to human labial salivary glands, acinar epithelial cells of the healthy control BALB/c mice contained two of the conventional PKC isoforms, alpha and beta. Acinar and ductal epithelial cells also contained the atypical PKC isoforms lambda and iota. PKC isoforms gamma, delta, epsilon, and theta were not found. NOD mice which displayed focal sialadenitis contained the same conventional and atypical PKC isoforms. The acinar cells in NOD mice, in contrast to the Sjögren's syndrome patients, did not lack PKC alpha or beta. On the contrary, PKC alpha and beta staining was stronger than in the control BALB/c mice. The present results demonstrate that both conventional and atypical PKC isoforms participate in the salivary epithelial cell biology and that there are mouse strain-associated and/or disease state-associated changes in their expression. The lack of PKC alpha and beta isoforms found in Sjögren's syndrome was not reproduced in NOD mice, which discloses one more difference between the human disease and its NOD mouse model.

  16. 鼻中隔涎腺腺泡细胞癌1例%Salivary gland acinar cell carcinoma at nasal septum:a case report

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Salivary gland acinar cell carcinoma is a rare low grade malignant tumor,which always occurs in the parotid,submandibular and sublingual salivary glands,but extremely rare occurs in the nasal septum.This paper reports a case of the salivary gland acinar cell carcinoma that located in the nasal septum.%涎腺腺泡细胞癌是一种临床上少见的低度恶性肿瘤,多发生于腮腺、颌下腺、舌下腺及小唾液腺等涎腺。发生于鼻中隔的极为罕见,本文报道一例近期发现的位于鼻中隔的涎腺腺泡细胞癌。

  17. Mitochondrial targeted β-lapachone induces mitochondrial dysfunction and catastrophic vacuolization in cancer cells.

    Science.gov (United States)

    Ma, Jing; Lim, Chaemin; Sacher, Joshua R; Van Houten, Bennett; Qian, Wei; Wipf, Peter

    2015-11-01

    Mitochondria play important roles in tumor cell physiology and survival by providing energy and metabolites for proliferation and metastasis. As part of their oncogenic status, cancer cells frequently produce increased levels of mitochondrial-generated reactive oxygen species (ROS). However, extensive stimulation of ROS generation in mitochondria has been shown to be able to induce cancer cell death, and is one of the major mechanisms of action of many anticancer agents. We hypothesized that enhancing mitochondrial ROS generation through direct targeting of a ROS generator into mitochondria will exhibit tumor cell selectivity, as well as high efficacy in inducing cancer cell death. We thus synthesized a mitochondrial targeted version of β-lapachone (XJB-Lapachone) based on our XJB mitochondrial targeting platform. We found that the mitochondrial targeted β-lapachone is more efficient in inducing apoptosis compared to unconjugated β-lapachone, and the tumor cell selectivity is maintained. XJB-Lapachone also induced extensive cellular vacuolization and autophagy at a concentration not observed with unconjugated β-lapachone. Through characterization of mitochondrial function we revealed that XJB-Lapachone is indeed more capable of stimulating ROS generation in mitochondria, which led to a dramatic mitochondrial uncoupling and autophagic degradation of mitochondria. Taken together, we have demonstrated that targeting β-lapachone accomplishes higher efficacy through inducing ROS generation directly in mitochondria, resulting in extensive mitochondrial and cellular damage. XJB-Lapachone will thus help to establish a novel platform for the design of next generation mitochondrial targeted ROS generators for cancer therapy.

  18. Leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of prostaglandin and constitutive nitric oxide synthase pathways.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2008-04-01

    Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE2 generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE2. The leptin-induced changes in arachidonic acid release and PGE2 generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

  19. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient.

    Science.gov (United States)

    Pfrommer, Sarah; Weber, Achim; Dutkowski, Philipp; Schäfer, Niklaus G; Müllhaupt, Beat; Bourquin, Jean-Pierre; Breitenstein, Stefan; Pestalozzi, Bernhard C; Stenner, Frank; Renner, Christoph; D'Addario, Giannicola; Graf, Hans-Jörg; Knuth, Alexander; Clavien, Pierre-Alain; Samaras, Panagiotis

    2013-01-01

    Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life. PMID:24163668

  20. Successful Salvage Chemotherapy with FOLFIRINOX for Recurrent Mixed Acinar Cell Carcinoma and Ductal Adenocarcinoma of the Pancreas in an Adolescent Patient

    Directory of Open Access Journals (Sweden)

    Sarah Pfrommer

    2013-09-01

    Full Text Available Pancreatic tumors are rare in children and adolescents. Here, we report the case of a 15-year-old boy who presented with a mixed acinar cell carcinoma/ductal adenocarcinoma with blastomatous components. He received multimodal treatment including various chemotherapy regimens and multistep surgery including liver transplantation. Introduction of FOLFIRINOX after relapse repeatedly achieved a durable metabolic and clinical response with good quality of life.

  1. Pancreatic panniculitis in a patient with pancreatic-type acinar cell carcinoma of the liver – case report and review of literature

    OpenAIRE

    Zundler, Sebastian; Erber, Ramona; Agaimy, Abbas; Hartmann, Arndt; Kiesewetter, Franklin; Strobel, Deike; Neurath, Markus F; Wildner, Dane

    2016-01-01

    Background Pancreatic panniculitis is a rare condition, which has only been described in relation with pancreatic diseases up to now. It is characterized by necrotizing subcutaneous inflammation and is thought to be triggered by adipocyte necrosis due to systemic release of pancreatic enzymes with consecutive infiltration of neutrophils. We present the first case of a patient with pancreatic panniculitis caused by pancreatic-type primary acinar cell carcinoma (ACC) of the liver and without un...

  2. The natural history of pancreatic acinar cell cystadenoma: Is resection better than surveillance? An update to a case report from 2010

    OpenAIRE

    Darcy, David G.; Dominique Jan

    2016-01-01

    Cystic lesions of the pancreas are a rare entity, and few reports have described their natural history in children. A previously published report described a 9-year-old boy with an acinar cell cystadenoma, discovered during a laparoscopic appendectomy. Initially asymptomatic and followed by serial MRI, this patient presented to our institution several years later with chronic obstructive symptoms that required surgical intervention. Planning for resection included multidisciplinary input from...

  3. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition.

    Science.gov (United States)

    Ram, Babul Moni; Ramakrishna, Gayatri

    2014-11-01

    Cyclosporine A (CsA), a widely used immunosuppressant shows cytotoxic effects by either inducing apoptosis or redirecting the cell towards non-apoptotic cell death. However, there still remains a lacuna in understanding the mechanism of CsA induced non-apoptotic cell death. In the present study we investigated calcineurin dependent or independent cytotoxic effects of CsA, a calcineurin inhibitor, in cervical cancerous SiHa cells. Decreased cell viability and massive cytoplasmic vacuolations were observed in CsA treated SiHa cells, having increased calcineurin activity. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR), accompanied by a decrease in cyclophilin B (ER resident PPIase), preceded the formation of the vacuoles. These vacuoles stained positive for many ER resident markers confirming their ER origin; but the absence of autophagosomal marker, LC3II, ruled out autophagy. Extensively vacuolated cells eventually undergo cell death which lacked the typical apoptotic features, but showed significant decrease in AIP (ALG2 interacting protein) as seen in paraptosis. ER-vacuolation was prevented by cycloheximide and salubrinal thereby indicating requirement of active protein synthesis. Inhibiting calcineurin activity by either Tacrolimus (FK506) or by knockdown of calcineurin B subunit did not result in either ER-stress or cellular vacuolation. However, knockdown of cyclophilin B by siRNA resulted in increased expression of Bip and IRE1α, together with cytoplasmic vacuolation. In conclusion, we report that persistent ER stress due to cyclophilin B inhibition in CsA treated cervical cancer cells caused cellular vacuolation which culminated in a non-apoptotic cell death response similar to paraptosis. Additionally, the paraptotic effects of CsA are independent of calcineurin inhibition. PMID:25003316

  4. E-cadherin-negative acinar cell carcinoma of the pancreas: report of a case showing a solid pseudopapillary growth pattern.

    Science.gov (United States)

    Tajima, Shogo; Waki, Michihiko; Azuma, Masaki; Koda, Kenji; Ohata, Akihiko

    2016-09-01

    E-cadherin expression patterns in acinar cell carcinomas (ACCs) of the pancreas have not been well documented. Herein, we present a hitherto undescribed case of E-cadherin-negative ACC with a solid pseudopapillary growth pattern in a 65-year-old man. We used an antibody against the extracellular domain of E-cadherin. As a further unusual status in ACC, faint β-catenin expression was observed in the cytoplasm of carcinoma cells. Morphological distinction from a solid pseudopapillary neoplasm (SPN) of the pancreas might be problematic in such a case, because of their similarities concerned with the growth pattern and E-cadherin negativity. Without nuclear accumulation of β-catenin, a diagnosis of SPN was almost excluded. Immunoreactivity for trypsin and BCL10 made an accurate diagnosis of ACC to this case. The tumor recurred 10 months post-surgery as rapidly enlarging masses in the liver, presumably indicating the aggressiveness of the E-cadherin-negative phenotype among ACCs. PMID:25600280

  5. SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats.

    Science.gov (United States)

    Sabino-Silva, Robinson; Alves-Wagner, Ana B T; Burgi, Katia; Okamoto, Maristela M; Alves, Adilson S; Lima, Guilherme A; Freitas, Helayne S; Antunes, Vagner R; Machado, Ubiratan F

    2010-12-01

    Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (∼30%, P acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

  6. Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

    Directory of Open Access Journals (Sweden)

    F D’Amico

    2009-12-01

    Full Text Available The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA, Ulex europaeus agglutinin I (UEA-I, Glycine max agglutinin (SBA, Arachys hypogaea agglutinin (PNA]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory gran- ules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.

  7. CT and MR imaging of multilocular acinar cell cystadenoma: comparison with branch duct intraductal papillary mucinous neoplasia (IPMNs)

    Energy Technology Data Exchange (ETDEWEB)

    Delavaud, Christophe; Assignies, Gaspard d' ; Vilgrain, Valerie; Vullierme, Marie-Pierre [Hopital Beaujon, Service de Radiologie, Clichy (France); Cros, Jerome [Hopital Beaujon, Service d' Anatomopathologie, Clichy (France); Ruszniewski, Philippe; Hammel, Pascal; Levy, Philippe [Hopital Beaujon, Service de Pancreato-Gastro-Enterologie, Clichy (France); Couvelard, Anne [Hopital Bichat, Service d' Anatomopathologie, Paris (France); Sauvanet, Alain; Dokmak, Safi [Hopital Beaujon, Service de Chirurgie Hepato-Pancreato-Biliaire, Clichy (France)

    2014-09-15

    To describe CT and MR imaging findings of acinar cell cystadenoma (ACC) of the pancreas and to compare them with those of branch duct intraductal papillary mucinous neoplasia (BD-IPMN) to identify distinctive elements. Five patients with ACC and the 20 consecutive patients with histologically proven BD-IPMN were retrospectively included. Clinical and biological information was collected and histological data reviewed. CT and MR findings were analysed blinded to pathological diagnosis in order to identify imaging diagnostic criteria of ACC. Patients with ACC were symptomatic in all but one case and were younger than those with BD-IPMN (p = 0.006). Four radiological criteria allowed for differentiating ACC from IPMN: five or more cysts, clustered peripheral small cysts, presence of cyst calcifications and absence of communication with the main pancreatic duct (p < 0.05). Presence of at least two or three of these imaging criteria had a strong diagnostic value for ACC with a sensitivity of 100 % and 80 % and a specificity of 85 % and 100 %, respectively. Preoperative differential diagnosis between ACC and BD-IPMN can be achieved using a combination of four CT and/or MR imaging criteria. Recognition of ACC patients could change patient management and lead to more conservative treatment. (orig.)

  8. Delivery of prolamins to the protein storage vacuole in maize aleurone cells.

    Science.gov (United States)

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

    2011-02-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-15

    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. The 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.

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

    International Nuclear Information System (INIS)

    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. The 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

  11. Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation.

    Science.gov (United States)

    Tateishi, Yoshihisa; Sasabe, Eri; Ueta, Eisaku; Yamamoto, Tetsuya

    2008-02-01

    Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by gamma-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.

  12. Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells.

    Science.gov (United States)

    Guyer, Debra M; Radulovic, Suzana; Jones, Faye-Ellen; Mobley, Harry L T

    2002-08-01

    The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

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

    Directory of Open Access Journals (Sweden)

    Noriaki Kadohama

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  16. Characteristics and mechanism of enzyme secretion and increase in [ Ca2+ ]i in Saikosaponin(I) stimulated rat pancreatic acinar cells

    Institute of Scientific and Technical Information of China (English)

    Yi Yu; Wen-Xiu Yang; Hui Wang; Wen-Zheng Zhang; Bao-Hua Liu; Zhi-Yong Dong

    2002-01-01

    AIM: This investigation was to reveal the characteristics andmechanism of enzyme secretion and increase in [ Ca2+ ]1stimulated by saikosaponin(I) [ SA(I) ] in rat pancreatic acini.METHODS: Pancreatic acini were prepared from male Wistarrats. Isolated acinar cells were suspended in Eagle's MEMsolution. After adding drugs, the incubation was performedat 37 ℃ for a set period of time. Amylase of supernatant wasassayed using starch-iodide reaction. Isolated acinar singlecell was incubated with Fura-2/AMat 37 ℃, then cells werewashed and resuspended in fresh solution and attached tothe chamber. Cytoplasm [ Ca2+ ]i of a single cell wasexpressed by fluorescence ratio F340/F380 recorded in aNikon PI Ca2+ measurement system.RESULTS: Rate course of amylase secretion stimulated bySA(I) in rat pancreatic acini appeared in bell-like shape. Thepeak amplitude increased depended on SA(I) concentration.The maximum rate responded to 1 × 10-5 mol/L SA(I) was 13.1-fold of basal and the rate decreased to basal level at 30min. CCK-8 receptor antagonist Bt2-cGMP markedly inhibitedamylase secretion stimulated by SA (I) and the dose-effectrelationship was similar to that by CCK-8. [Ca2+ ]i in a singleacinar cell rose to the peak st 5 min after adding 5 × l06 mol/LSA(I) and was 5. 1-fold of basal level. In addition, there was asecondary increase after the initial peak. GDP could inhibitboth the rate of amylase secretion and rising of [Ca2+ ]istimulated by SA(I) in a single pancreatic acinar cell.CONCLUSION: SA (I) is highly efficient in promoting thesecretion of enzymes synthesized in rat pancreatic acini andraising intracellular [Ca2+ ]i. Signaling transduction pathwayof SA(I) involves activating special membrane receptor andincrease in cytoplasm [Ca2+ ]i sequentially.

  17. Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells.

    Science.gov (United States)

    Park, Hyung Seo; Betzenhauser, Matthew J; Zhang, Yu; Yule, David I

    2012-01-01

    Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 μM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

  18. Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin.

    Science.gov (United States)

    Papini, E; Satin, B; Norais, N; de Bernard, M; Telford, J L; Rappuoli, R; Montecucco, C

    1998-01-01

    The effects of the vacuolating toxin (VacA) released by pathogenic strains of Helicobacter pylori on several polarized epithelial monolayers were investigated. Trans-epithelial electric resistance (TER) of monolayers formed by canine kidney MDCK I, human gut T84, and murine mammary gland epH4, was lowered by acid-activated VacA. Independent of the cell type and of the starting TER value, VacA reduced it to a minimal value of 1,000-1,300 Omega x cm2. TER decrease was paralleled by a three- to fourfold increase of [14C]-mannitol (molecular weight 182.2) and a twofold increase of [14C]-sucrose (molecular weight 342.3) transmonolayer flux. On the contrary, transmembrane flux of the proinflammatory model tripeptide [14C]-N-formyl-Met-Leu-Phe (molecular weight 437.6), of [3H]-inuline (molecular weight 5,000) and of HRP (molecular weight 47,000) did not change. These data indicate that VacA increases paracellular epithelial permeability to molecules with molecular weight < 350-440. Accordingly, the epithelial permeability of Fe3+ and Ni2+ ions, essential for H. pylori survival in vivo, was also increased by VacA. High-resolution immunofluorescence and SDS-PAGE analysis failed to reveal alterations of junctional proteins ZO-1, occludin, cingulin, and E-cadherin. It is proposed that induction by VacA of a selective permeabilization of the epithelial paracellular route to low molecular weight molecules and ions may serve to supply nutrients, which favor H. pylori growth in vivo. PMID:9710450

  19. Rapid structural changes and acidification of guard cell vacuoles during stomatal closure require phosphatidylinositol 3,5-bisphosphate.

    Science.gov (United States)

    Bak, Gwangbae; Lee, Eun-Jung; Lee, Yuree; Kato, Mariko; Segami, Shoji; Sze, Heven; Maeshima, Masayoshi; Hwang, Jae-Ung; Lee, Youngsook

    2013-06-01

    Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)-induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H(+)-ATPase vha-a2 vha-a3 and vacuolar H(+)-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution. PMID:23757398

  20. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

    Science.gov (United States)

    Marciniak, Anja; Cohrs, Christian M; Tsata, Vasiliki; Chouinard, Julie A; Selck, Claudia; Stertmann, Julia; Reichelt, Saskia; Rose, Tobias; Ehehalt, Florian; Weitz, Jürgen; Solimena, Michele; Slak Rupnik, Marjan; Speier, Stephan

    2014-12-01

    Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

  1. Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome.

    Science.gov (United States)

    Hauk, V; Calafat, M; Larocca, L; Fraccaroli, L; Grasso, E; Ramhorst, R; Leirós, C Pérez

    2011-12-01

    Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non-obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti-inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)-α and reversed by VIP through a cyclic adenosine-5'-monophosphate (cAMP)/protein kinase A (PKA)-mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss.

  2. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS. We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  3. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells.

    Science.gov (United States)

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS). We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

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

    Energy Technology Data Exchange (ETDEWEB)

    Loo Jr., Billy W.

    2000-06-09

    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 major 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.

  5. Glucose alleviates cadmium toxicity by increasing cadmium fixation in root cell wall and sequestration into vacuole in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yuan-Zhi Shi; Xiao-Fang Zhu; Jiang-Xue Wan; Gui-Xin Li; Shao-Jian Zheng

    2015-01-01

    Glucose (Glu) is involved in not only plant physiological and developmental events but also plant responses to abiotic stresses. Here, we found that the exogenous Glu improved root and shoot growth, reduced shoot cadmium (Cd) concentration, and rescued Cd-induced chlorosis in Arabidopsis thaliana (Columbia ecotype, Col-0) under Cd stressed conditions. Glucose increased Cd retained in the roots, thus reducing its translocation from root to shoot significantly. The most Cd retained in the roots was found in the hemicellulose 1. Glucose combined with Cd (Glu þ Cd) treatment did not affect the content of pectin and its binding capacity of Cd while it increased the content of hemicelluloses 1 and the amount of Cd retained in it significantly. Furthermore, Leadmium Green staining indicated that more Cd was compartmented into vacuoles in Glu þ Cd treatment compared with Cd treatment alone, which was in accordance with the significant upregulation of the expression of tonoplast-localized metal transporter genes, suggesting that com-partmentation of Cd into vacuoles also contributes to the Glu-alleviated Cd toxicity. Taken together, we demonstrated that Glu-alleviated Cd toxicity is mediated through increas-ing Cd fixation in the root cell wall and sequestration into the vacuoles.

  6. Human lung epithelial cells contain Mycobacterium tuberculosis in a late endosomal vacuole and are efficiently recognized by CD8⁺ T cells.

    Directory of Open Access Journals (Sweden)

    Melanie J Harriff

    Full Text Available Mycobacterium tuberculosis (Mtb is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells. Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.

  7. Impact of PI3Kγ gene knockout on acinar cells in mice with acute pancreatitis%磷脂酰肌醇3-激酶γ基因敲除对急性胰腺炎小鼠腺泡细胞的影响

    Institute of Scientific and Technical Information of China (English)

    贾文焯; 孙建华; 余涛; 肖刚

    2012-01-01

    injections of cerulean and mice of the control group were subjected to exactly the same regimen of saline injections. In addition, the pancreatic acini were isolated from another two different sets of mice (8 per set), and then the acinar cells were stimulated with CCK-8 to prepare an in vitro AP model. Control cells were treated with DMSO instead of CCK-8. Pathological changes of the pancreatic tissues were assessed, and the serum level of amylase, trypsin activity in pancreatic tissues and acinar cells, and level amylase release from the acinar cells were measured. The HSP70 protein expressions in pancreatic tissues and acinar cells were determined by Western blot analysis.Results: In pathological observation, the pancreatic tissues from the control groups of both types of mice showed no abnormality, while both AP groups presented varying degrees of edema, necrosis and hemorrhage. The quantitative analysis showed that the number of necrotic acinar cells and vacuoles of the KO mice were significantly less than those of the WT mice (both P0.05), but the trypsin activity of the pancreatic tissues and isolated acinar cells of the KO mice were significantly lower than those of the WT mice in the AP groups (in vivo and in vitro) (both P0.05). Compared with the control groups, the HSP70 protein expressions in both pancreatic tissues and acinar cells increased obviously in AP groups (in vivo and in vitro), in which the HSP70 expression levels of the KO mice were significantly higher than those of the WT mice (both P<0.05).Conclusion: PI3Kγ may promote acinar necrosis in acute pancreatitis by down-regulating HSP70 protein expressions and enhancing activation of trypsinogen, but it has no obvious effect on amylase secretion.

  8. Effects of a diet high in fish oil (MaxEPA) on the formation of micronucleated erythrocytes in blood and on the number of atypical acinar cell foci induced in rat pancreas by Azaserine

    NARCIS (Netherlands)

    Appel, M.J.; Woutersen, R.A.

    2004-01-01

    The present study was performed to investigate the influence of fish oil on the genotoxic effects of azaserine, using the formation of micronucleated erythrocytes as a measure for the degree of initiating potency and the number and size of putative preneoplastic pancreatic atypical acinar cell foci

  9. Fractionated irradiation and late changes in rat parotid gland: effects on the number of acinar cells, potassium efflux, and amylase secretion

    Energy Technology Data Exchange (ETDEWEB)

    Franzen, L.; Gustafsson, H.; Sundstroem, S.; Karlsson, M.; Littbrand, B.; Henriksson, R. (Umeaa Univ. Hospital (Sweden). Dept. of Oncology, Otorhinolaryngology, Histology and Cell Biology)

    1993-07-01

    The authors used different in vitro secretory models and quantitative morphological characterization of rat parotid gland following fractionated unilateral irradiation to one gland on a 5-day fraction schedule with 6 MV photons (total dose 30, 35, 40 and 45 Gy) or a two-fractions regimen in 5 days with total dose of 24 and 32 Gy. The contralateral shielded gland served as control, and parallel analyses of irradiated and control glands were performed 180 days following the last irradiation. The relative noradrenaline stimulated electrolyte secretion ([sup 86]rubidium tracer for potassium) was decreased in the irradiated compared with control glands. The noradrenaline-stimulated exocytotic amylase release was not significantly affected by irradiation, but the gland content of amylase was decreased dose-dependently. The quantitative morphological analysis revealed a dose-dependent decline in the number of acinar cells; the other parenchymal cells were unaffected by irradiation compared with controls. (author).

  10. Fractionated irradiation and late changes in rat parotid gland: effects on the number of acinar cells, potassium efflux, and amylase secretion

    International Nuclear Information System (INIS)

    The authors used different in vitro secretory models and quantitative morphological characterization of rat parotid gland following fractionated unilateral irradiation to one gland on a 5-day fraction schedule with 6 MV photons (total dose 30, 35, 40 and 45 Gy) or a two-fractions regimen in 5 days with total dose of 24 and 32 Gy. The contralateral shielded gland served as control, and parallel analyses of irradiated and control glands were performed 180 days following the last irradiation. The relative noradrenaline stimulated electrolyte secretion (86rubidium tracer for potassium) was decreased in the irradiated compared with control glands. The noradrenaline-stimulated exocytotic amylase release was not significantly affected by irradiation, but the gland content of amylase was decreased dose-dependently. The quantitative morphological analysis revealed a dose-dependent decline in the number of acinar cells; the other parenchymal cells were unaffected by irradiation compared with controls. (author)

  11. Slug inhibits pancreatic cancer initiation by blocking Kras-induced acinar-ductal metaplasia

    OpenAIRE

    Kazumi Ebine; Chow, Christina R.; DeCant, Brian T.; Hattaway, Holly Z.; Grippo, Paul J.; Krishan Kumar; Munshi, Hidayatullah G.

    2016-01-01

    Cells in the pancreas that have undergone acinar-ductal metaplasia (ADM) can transform into premalignant cells that can eventually become cancerous. Although the epithelial-mesenchymal transition regulator Snail (Snai1) can cooperate with Kras in acinar cells to enhance ADM development, the contribution of Snail-related protein Slug (Snai2) to ADM development is not known. Thus, transgenic mice expressing Slug and Kras in acinar cells were generated. Surprisingly, Slug attenuated Kras-induced...

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

    Science.gov (United States)

    Lee, Jingu; Park, Sangkyu; Roh, Sangho

    2015-05-15

    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. The 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.

  13. Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells

    Directory of Open Access Journals (Sweden)

    Verena Ibl

    2014-09-01

    Full Text Available The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.

  14. Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells.

    Science.gov (United States)

    Ibl, Verena; Stoger, Eva

    2014-01-01

    The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs) in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.

  15. Direct imaging of RAB27B-enriched secretory vesicle biogenesis in lacrimal acinar cells reveals origins on a nascent vesicle budding site.

    Directory of Open Access Journals (Sweden)

    Lilian Chiang

    Full Text Available This study uses YFP-tagged Rab27b expression in rabbit lacrimal gland acinar cells, which are polarized secretory epithelial cells, to characterize early stages of secretory vesicle trafficking. Here we demonstrate the utility of YFP-Rab27b to delineate new perspectives on the mechanisms of early vesicle biogenesis in lacrimal gland acinar cells, where information is significantly limited. Protocols were developed to deplete the mature YFP-Rab27b-enriched secretory vesicle pool in the subapical region of the cell, and confocal fluorescence microscopy was used to track vesicle replenishment. This analysis revealed a basally-localized organelle, which we termed the "nascent vesicle site," from which nascent vesicles appeared to emerge. Subapical vesicular YFP-Rab27b was co-localized with p150(Glued, a component of the dynactin cofactor of cytoplasmic dynein. Treatment with the microtubule-targeted agent, nocodazole, did not affect release of mature secretory vesicles, although during vesicle repletion it significantly altered nascent YFP-Rab27b-enriched secretory vesicle localization. Instead of moving to the subapical region, these vesicles were trapped at the nascent vesicle site which was adjacent to, if not a sub-compartment of, the trans-Golgi network. Finally, YFP-Rab27b-enriched secretory vesicles which reached the subapical cytoplasm appeared to acquire the actin-based motor protein, Myosin 5C. Our findings show that Rab27b enrichment occurs early in secretory vesicle formation, that secretory vesicles bud from a visually discernable nascent vesicle site, and that transport from the nascent vesicle site to the subapical region requires intact microtubules.

  16. Effects of the disaggregation of high-polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture (stomata opening)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Observation under an electron microscope reveals that in closed and open stomata of V. faba, the average volume of particles in guard cell vacuoles (GCV) reduces about 3 orders in magnitude, while the distribution density of the particles increases about 2 orders of magnitude. By using the method of the ratio of fluorescent emissions with laser scanning confocal microscopy, the monitoring to stomata opening shows that during 10 to 30 s before the first distinguishable aperture of stomata, there is a change of pH in GCV about-0.5 units. A quick stomatal opening immediately follows the changes of pH in GCV to reach a steady aperture about 12μm in 100-200 s. This work proposes a model for the osmoregulation in GCV for stomatal opening. The proposed osmoregulation is related to the disaggregation of some polymerized particles inside GCV, which is probably induced by a -(pH in the vacuole. This model describes a process of osmoregulation that avoids the massive energy consuming transportation across cell membranes, which is a foundation of the current chemiosmotic hypothesis. This model is a supplement to the multiple controlling hypothesis for the stomatal movement, which widens research principle ideas for other quick movements in plants.

  17. Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2010-06-01

    Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

  18. Role of epidermal growth factor receptor transactivation in the activation of cytosolic phospholipase A(2) in leptin protection of salivary gland acinar cells against ethanol cytotoxicity.

    Science.gov (United States)

    Slomiany, B L; Slomiany, A

    2009-06-01

    A pleiotropic hormone, leptin, secreted into saliva by the acinar cells of salivary glands is an important mediator of the processes of oral mucosal defense. Here, we report on the role of epidermal growth factor receptor (EGFR) transactivation in the signaling events that mediate leptin protection of sublingual salivary gland acinar cells against ethanol cytotoxicity. We show that the protective effect of leptin against ethanol cytotoxicity was associated with the increased EGFR protein tyrosine kinase and cytosolic phospholipase A(2) (cPLA(2)) activity, and characterized by a marked increase in matrix metalloproteinase MMP-9 and arachidonic acid (AA) release, and PGE(2) generation. The loss in countering capacity of leptin against ethanol cytotoxicity was attained with JAK inhibitor AG490, Src inhibitor PP2, and EGFR inhibitor AG1478, as well as ERK inhibitor PD98059. Moreover, the agents evoked also the inhibition in leptin-induced up-regulation in cPLA(2) activity, AA release, and PGE(2) generation. The changes caused by leptin in EGFR phosphorylation, MMP-9, and cPLA(2) activation were susceptible to suppression by metalloprotease inhibitor GM6001, but the production of MMP-9 was not affected by EGFR inhibitor AG1478 or PKC inhibitor Ro318220. These findings point to the involvement of MMP-9 in the event of leptin-induced EGFR transactivation that results in the signaling cascade leading to cPLA(2) activation and up-regulation in PGE(2) generation, thus providing new insights into the mechanism of oral mucosal protection against ethanol toxicity.

  19. Adult pancreatic acinar cells give rise to ducts but not endocrine cells in response to growth factor signaling

    OpenAIRE

    Blaine, Stacy A.; Ray, Kevin C.; Anunobi, Reginald; Gannon, Maureen A.; Washington, Mary K.; Means, Anna L.

    2010-01-01

    Studies in both humans and rodents have found that insulin+ cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin+ cells arise de novo from ductal epithelium. We have found that insulin+ cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their...

  20. Melatonin induces the expression of Nrf2-regulated antioxidant enzymes via PKC and Ca2+ influx activation in mouse pancreatic acinar cells.

    Science.gov (United States)

    Santofimia-Castaño, Patricia; Clea Ruy, Deborah; Garcia-Sanchez, Lourdes; Jimenez-Blasco, Daniel; Fernandez-Bermejo, Miguel; Bolaños, Juan P; Salido, Gines M; Gonzalez, Antonio

    2015-10-01

    The goal of this study was to evaluate the potential activation of the nuclear factor erythroid 2-related factor and the antioxidant-responsive element (Nrf2-ARE) signaling pathway in response to melatonin in isolated mouse pancreatic acinar cells. Changes in intracellular free Ca(2+) concentration were followed by fluorimetric analysis of fura-2-loaded cells. The activations of PKC and JNK were measured by Western blot analysis. Quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of Nrf2-regulated antioxidant enzymes. Immunocytochemistry was employed to determine nuclear location of phosphorylated Nrf2, and the cellular redox state was monitored following MitoSOX Red-derived fluorescence. Our results show that stimulation of fura-2-loaded cells with melatonin (1 µM to 1 mM), in the presence of Ca(2+) in the extracellular medium, induced a slow and progressive increase of [Ca(2+)](c) toward a stable level. Melatonin did not inhibit the typical Ca(2+) response induced by CCK-8 (1 nM). When the cells were challenged with indoleamine in the absence of Ca(2+) in the extracellular solution (medium containing 0.5 mM EGTA) or in the presence of 1 mM LaCl(3), to inhibit Ca(2+) entry, we could not detect any change in [Ca(2+)](c). Nevertheless, CCK-8 (1 nM) was able to induce the typical mobilization of Ca(2+). When the cells were incubated with the PKC activator PMA (1 µM) in the presence of Ca(2+) in the extracellular medium, we observed a response similar to that noted when the cells were challenged with melatonin 100 µM. However, in the presence of Ro31-8220 (3 µM), a PKC inhibitor, stimulation of cells with melatonin failed to evoke changes in [Ca(2+)]c. Immunoblots, using an antibody specific for phospho-PKC, revealed that melatonin induces PKCα activation, either in the presence or in the absence of external Ca(2+). Melatonin induced the phosphorylation and nuclear translocation of the transcription factor Nrf2, and

  1. Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

    Science.gov (United States)

    Silva, Victor Diogenes A; Pitanga, Bruno P S; Nascimento, Ravena P; Souza, Cleide S; Coelho, Paulo Lucas C; Menezes-Filho, Noélio; Silva, André Mário M; Costa, Maria de Fátima D; El-Bachá, Ramon S; Velozo, Eudes S; Costa, Silvia L

    2013-12-16

    Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 μg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 μg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 μg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in β-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

  2. Knockdown of GRP78 promotes apoptosis in pancreatic acinar cells and attenuates the severity of cerulein and LPS induced pancreatic inflammation.

    Directory of Open Access Journals (Sweden)

    Yong Liu

    Full Text Available Acute pancreatitis (AP is a potentially lethal disease characterized by inflammation and parenchymal cell death; also, the severity of AP correlates directly with necrosis and inversely with apoptosis. However, mechanisms of regulating cell death in AP remain unclear. The endoplasmic reticulum (ER chaperone protein GRP78 has anti-apoptotic properties, in addition to modulating ER stress responses. This study used RNA interference (RNAi approach to investigate the potential role of GRP78 in regulating apoptosis during AP. In vitro models of AP were successfully developed by treating AR42J cells with cerulein or cerulein plus lipoplysaccharide (LPS. There was more pancreatic inflammation and less apoptosis with the cerulein plus LPS treatment. Furthermore, knockdown of GRP78 expression markedly promoted apoptosis and reduced necrosis in pancreatic acinar cells. This was accomplished by enhancing the activation of caspases and inhibiting the activity of X-linked inhibitor of apoptosis protein (XIAP, as well as a receptor interacting protein kinase-1(RIPK1, which is a key mediator of necrosis. This attenuated the severity of pancreatic inflammation, especially after cerulein plus LPS treatment. In conclusion, these findings indicate that GRP78 plays an anti-apoptotic role in regulating the cell death response during AP. Therefore, GRP78 is a potential therapeutic target for AP.

  3. Pancreatic ductal bicarbonate secretion: challenge of the acinar acid load

    Directory of Open Access Journals (Sweden)

    Peter eHegyi

    2011-07-01

    Full Text Available Acinar and ductal cells of the exocrine pancreas form a close functional unit. Although most studies contain data either on acinar or ductal cells, an increasing number of evidence highlights the importance of the pancreatic acinar-ductal functional unit. One of the best examples for this functional unit is the regulation of luminal pH by both cell types. Protons co-released during exocytosis from acini cause significant acidosis, whereas, bicarbonate secreted by ductal cells cause alkalization in the lumen. This suggests that the first and probably one of the most important role of bicarbonate secretion by pancreatic ductal cells is not only to neutralize the acid chyme entering into the duodenum from the stomach, but to neutralize acidic content secreted by acinar cells. To accomplish this role, it is more than likely that ductal cells have physiological sensing mechanisms which would allow them to regulate luminal pH. To date, four different classes of acid-sensing ion channels have been identified in the gastrointestinal tract (transient receptor potential ion channels, two-pore domain potassium channel, ionotropic purinoceptor and acid-sensing ion channel, however, none of these have been studied in pancreatic ductal cells. In this mini-review, we summarize our current knowledge of these channels and urge scientists to characterize ductal acid-sensing mechanisms and also to investigate the challenge of the acinar acid load on ductal cells.

  4. Vacuole Partitioning during Meiotic Division in Yeast

    OpenAIRE

    Roeder, A D; Shaw, J.M.

    1996-01-01

    We have examined the partitioning of the yeast vacuole during meiotic division. In pulse-chase experiments, vacuoles labeled with the lumenal ade2 fluorophore or the membrane-specific dye FM 4-64 were not inherited by haploid spores. Instead, these fluorescent markers were excluded from spores and trapped between the spore cell walls and the ascus. Serial optical sections using a confocal microscope confirmed that spores did not inherit detectable amounts of fluorescently labeled vacuoles. Mo...

  5. Targeting host syntaxin-5 preferentially blocks Leishmania parasitophorous vacuole development in infected cells and limits experimental Leishmania infections.

    Science.gov (United States)

    Canton, Johnathan; Kima, Peter E

    2012-10-01

    Our previous observations established a role for syntaxin-5 in the development of Leishmania parasitophorous vacuoles (LPVs). In this study, we took advantage of the recent identification of Retro-2, a small organic molecule that can cause the redistribution of syntaxin-5; we show herein that Retro-2 blocks LPV development within 2 hours of adding it to cells infected with Leishmania amazonensis. In infected cells incubated for 48 hours with Retro-2, LPV development was significantly limited; furthermore, infected cells harbored four to five times fewer parasites than infected cells incubated in vehicle alone. In vivo studies revealed that Retro-2 curbed experimental L. amazonensis infections in a dose-dependent manner. Retro-2 did not have any appreciable effect on the host cell physiological characteristics; furthermore, it had no apparent toxicity in experimental animals. An unexpected, but welcome, finding was that Retro-2 inhibited the replication of Leishmania parasites in axenic cultures. This study is significant because it identifies an endoplasmic reticulum/Golgi SNARE as a potential target for the control of Leishmania infections; moreover, it suggests that small organic molecules can be identified that can selectively disrupt the vesicle fusion machinery that promotes the development of pathogen-containing compartments without exerting toxic effects on the host.

  6. Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells 1

    Science.gov (United States)

    Bouzayen, Mondher; Latché, Alain; Pech, Jean-Claude; Marigo, Gérard

    1989-01-01

    The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a Km of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H+ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN− and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH2— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions. PMID:16667182

  7. Tissue microarrays in pathological examination of apoptotic acinar cells induced by dexamethasone in the pancreas of rats with severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Xi-Ping Zhang; Hua Tian; Bei Lu; Li Chen; Ru-Jun Xu; Ke-Yi Wang; Zhi-Wei Wang; Qi-Hui Cheng; Hai-Ping Shen

    2007-01-01

    BACKGROUND:The good therapeutic effects of large dose of dexamethasone on severe acute pancreatitis (SAP) patients have been proved. This study was designed to investigate the inlfuence of dexamethasone on apoptosis of acinar cells in the pancreas of rats with SAP and the protein expression of the apoptosis-regulating genes Bax and Bcl-2. METHODS: Ninety Sprague-Dawley rats with SAP were randomly divided into a model group and a dexamethasone treated group (45 rats in each group), and another 45 rats formed the sham operation group. Survival rates were calculated and gross pathological changes in the pancreas of each group were observed under a light microscope 3, 6 and 12 hours after operation. Tissue microarray technology was applied to prepare pancreatic tissue sections. The changes in Bax and Bcl-2 protein expression levels of pancreatic tissues from each group were assessed by immunohistochemical staining, and TUNEL staining was used to evaluate changes in apoptosis index. RESULTS: The model and treated groups did not differ in mortality at each time point. The pathological score for the pancreas in the treated group was signiifcantly lower than that in the model group at 3 and 6 hours. The positive rates of Bax protein expression in the head and tail of the pancreas in the treated group at all time points were all markedly higher than those of the model group. The positive rate of Bcl-2 protein expression in the head of the pancreas in the treated group was signiifcantly higher than that of the model group at 3 hours. TUNEL staining showed that the pancreas head and tail apoptosis indices of the treated group were markedly higher than those of the model group after 6 hours. CONCLUSIONS: Apoptosis may be a protective response to pancreatic cell injury. The mechanism of action of dexamethasone in treating SAP may be related to the apoptosis of acinar cells in the pancreas induced by apoptosis-regulating genes such as Bax and Bcl-2. The advantages of tissue

  8. Serotonin promotes acinar dedifferentiation following pancreatitis-induced regeneration in the adult pancreas.

    Science.gov (United States)

    Saponara, Enrica; Grabliauskaite, Kamile; Bombardo, Marta; Buzzi, Raphael; Silva, Alberto B; Malagola, Ermanno; Tian, Yinghua; Hehl, Adrian B; Schraner, Elisabeth M; Seleznik, Gitta M; Zabel, Anja; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-12-01

    The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells.

  9. Serotonin promotes acinar dedifferentiation following pancreatitis-induced regeneration in the adult pancreas.

    Science.gov (United States)

    Saponara, Enrica; Grabliauskaite, Kamile; Bombardo, Marta; Buzzi, Raphael; Silva, Alberto B; Malagola, Ermanno; Tian, Yinghua; Hehl, Adrian B; Schraner, Elisabeth M; Seleznik, Gitta M; Zabel, Anja; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-12-01

    The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone-stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation-mediated tissue injury. Specifically, lack of serotonin resulted in delayed up-regulation of progenitor genes and delayed the formation of acinar-to-ductal metaplasia and defective acinar cell proliferation. We identified serotonin-dependent acinar secretion as a key step in progenitor-based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1-Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells. PMID:26235267

  10. Pancreatic Acinar Cells Employ miRNAs as Mediators of Intercellular Communication to Participate in the Regulation of Pancreatitis-Associated Macrophage Activation.

    Science.gov (United States)

    Zhao, Yong; Wang, Hao; Lu, Ming; Qiao, Xin; Sun, Bei; Zhang, Weihui; Xue, Dongbo

    2016-01-01

    Macrophage activation plays an important role in the inflammatory response in acute pancreatitis. In the present study, the activation of AR42J pancreatic acinar cells was induced by taurolithocholate treatment. The results showed that the culture medium from the activated AR42J cells significantly enhanced NFκB activation in the macrophages compared to that without taurolithocholate treatment. Additionally, the precipitates obtained from ultracentrifugation of the culture media that were rich in exosomes were markedly more potent in activating macrophages compared with the supernatant fraction lacking exosomes. The results indicated that the mediators carried by the exosomes played important roles in macrophage activation. Exosomal miRNAs were extracted and examined using microarrays. A total of 115 differentially expressed miRNAs were identified, and 30 showed upregulated expression, while 85 displayed downregulated expression. Target genes of the differentially expressed miRNAs were predicted using TargetScan, MiRanda, and PicTar software programs. The putative target genes were subjected to KEGG functional analysis. The functions of the target genes were primarily enriched in MAPK pathways. Specifically, the target genes regulated macrophage activation through the TRAF6-TAB2-TAK1-NIK/IKK-NFκB pathway. As the mediators of signal transduction, miRNAs and their predicted target mRNAs regulate every step in the MAPK pathway. PMID:27546996

  11. Vacuole formation in mast cells responding to osmotic stress and to F-actin disassembly

    DEFF Research Database (Denmark)

    Koffer, Anna; Williams, Mark; Johansen, Torben

    2002-01-01

    Fluorescent probes were used to visualize the morphology of membranes and of F-actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization...

  12. 青霉素处理检查和分离蓝藻细胞液泡%Determination and Isolation of Cell Vacuoles from Blue-green Algae by Penicillin Method

    Institute of Scientific and Technical Information of China (English)

    郭碧薇; 易平; 刘希玲; 郭厚良

    2003-01-01

    Growing in the liquid medium containing penicillin, the cells of the Cyanobacteria,Anabaena 7120, Nostoc flagelliforme, and Synechocystis 6803 were broken and vacuoles were released. Percentage of broken cells declined and percentage of broken cells increased with the growing days of the algae. The percentage of vacuoles to broken cells were respectively 0.7%, 0.8%, and 13.3% in the three types of algae Anabaena 7120, N.flagelliforme and Synechocystis 6803 which had grown for 3 days.

  13. Mevalonosomes: specific vacuoles containing the mevalonate pathway in Plocamium brasiliense cortical cells (Rhodophyta).

    Science.gov (United States)

    Paradas, Wladimir Costa; Crespo, Thalita Mendes; Salgado, Leonardo Tavares; de Andrade, Leonardo Rodrigues; Soares, Angélica Ribeiro; Hellio, Claire; Paranhos, Ricardo Rogers; Hill, Lilian Jorge; de Souza, Geysa Marinho; Kelecom, Alphonse Germaine Albert Charles; Da Gama, Bernardo Antônio Perez; Pereira, Renato Crespo; Amado-Filho, Gilberto Menezes

    2015-04-01

    This paper has identified, for the first time in a member of the Rhodophyta, a vacuolar organelle containing enzymes that are involved in the mevalonate pathway-an important step in red algal isoprenoid biosynthesis. These organelles were named mevalonosomes (Mev) and were found in the cortical cells (CC) of Plocamium brasiliense, a marine macroalgae that synthesizes several halogenated monoterpenes. P. brasiliense specimens were submitted to a cytochemical analysis of the activity of the 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS). Using transmission electron microscopy (TEM), we confirmed the presence of HMGS activity within the Mev. Because HMGS is necessary for the biosynthesis of halogenated monoterpenes, we isolated a hexanic fraction (HF) rich in halogenated monoterpenes from P. brasiliense that contained a pentachlorinated monoterpene as a major metabolite. Because terpenes are often related to chemical defense, the antifouling (AF) activity of pentachlorinated monoterpene was tested. We found that the settlement of the mussel Perna perna was reduced by HF treatment (2.25 times less than control; 40% and 90% of fouled surface, respectively; P = 0.001; F9,9 = 1.13). The HF (at 10 μg · mL(-1) ) also inhibited three species of fouling microalgae (Chlorarachnion reptans, Cylindrotheca cloisterium, and Exanthemachrysis gayraliae), while at a higher concentration (50 μg · mL(-1) ), it inhibited the bacteria Halomonas marina, Polaribacter irgensii, Pseudoalteromonas elyakovii, Shewanella putrefaciens, and Vibrio aestuarianus. The AF activity of P. brasiliense halogenated monoterpenes and the localization of HMGS activity inside Mev suggest that this cellular structure found in CC may play a role in thallus protection against biofouling. PMID:26986518

  14. Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate[C][W

    Science.gov (United States)

    Bak, Gwangbae; Lee, Eun-Jung; Lee, Yuree; Kato, Mariko; Segami, Shoji; Sze, Heven; Maeshima, Masayoshi; Hwang, Jae-Ung; Lee, Youngsook

    2013-01-01

    Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H+-ATPase vha-a2 vha-a3 and vacuolar H+-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P2, which is essential for vacuolar acidification and convolution. PMID:23757398

  15. Anaplasma phagocytophilum APH0032 Is Exposed on the Cytosolic Face of the Pathogen-Occupied Vacuole and Co-opts Host Cell SUMOylation

    Science.gov (United States)

    Oki, Aminat T.; Huang, Bernice; Beyer, Andrea R.; May, Levi J.; Truchan, Hilary K.; Walker, Naomi J.; Galloway, Nathan L.; Borjesson, Dori L.; Carlyon, Jason A.

    2016-01-01

    Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM's cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 in A. phagocytophilum infected host cells significantly boosted the bacterial load. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to

  16. Activation of ERK1/2 by store-operated calcium entry in rat parotid acinar cells.

    Directory of Open Access Journals (Sweden)

    Stephen P Soltoff

    Full Text Available The regulation of intracellular Ca(2+ concentration ([Ca(2+]i plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca(2+ stores, followed by the sustained elevation of [Ca(2+]i from Ca(2+ entry across the plasma membrane via store-operated Ca(2+ entry (SOCE. Ca(2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+ (ARC channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2 to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca(2+ stores using the endoplasmic reticulum Ca(2+-ATPase inhibitor thapsigargin (TG. TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca(2+]i by Ca(2+ ionophore or by Ca(2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB, SKF96363, and removal of extracellular Ca(2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate

  17. Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress.

    Science.gov (United States)

    Maathuis, F J; Prins, H B

    1990-01-01

    Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K) (+)/P(Na) (+) = 1, whereas the cation/anion selectivity (P(K) (+)/P(c1) (-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2)) = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.

  18. Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

    Science.gov (United States)

    Taylor, Michael J; Baicu, Simona C

    2014-01-01

    Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five 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 to distend the acinar; 3) freezing the entire Px solid to dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. 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 new and novel method that avoids the problems associated with conventional collagenase digestion methods. PMID:23992741

  19. Mechanism of Cytosolic Phospholipase A(2) Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity.

    Science.gov (United States)

    Slomiany, Bronislaw L; Slomiany, Amalia

    2010-01-01

    Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A(2) (cPLA(2)) activity and arachidonic acid (AA) release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA(2) activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA(2) phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA(2) protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA(2) activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

  20. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells in the hippocampal neurogenesis involving myelin vacuolation of cholinergic and glutamatergic inputs in mice.

    Science.gov (United States)

    Kato, Mizuho; Abe, Hajime; Itahashi, Megu; Kikuchihara, Yoh; Kimura, Masayuki; Mizukami, Sayaka; Yoshida, Toshinori; Shibutani, Makoto

    2016-02-01

    Hexachlorophene (HCP) has been shown to induce myelin vacuolation due to intramyelinic edema of the nerve fibers in animal neural tissue. We investigated the maternal exposure effect of HCP on hippocampal neurogenesis in the offspring of pregnant mice supplemented with 0 (control), 33 or 100 ppm HCP in diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, offspring as examined in males exhibited decreased granule cell lineage populations expressing paired box 6, sex-determining region Y-box 2 and eomesodermin in the hippocampal subgranular zone (SGZ) accompanied by myelin vacuolation involving white matter tracts of the hippocampal fimbria at ≥ 33 ppm. However, SGZ cellular populations expressing brain lipid binding protein and doublecortin were unchanged at any dose. Transcript expression of cholinergic receptor genes, Chrna4 and Chrnb2, and glutamate receptor genes, Grm1 and Grin2d, examined at 100 ppm, decreased in the dentate gyrus. HCP exposure did not alter the number of proliferating or apoptotic cells in the SGZ, or reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)ergic interneurons in the dentate hilus, on PND 21 and PND 77. All neurogenesis-related changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77, suggesting that maternal HCP exposure at ≥ 33 ppm reversibly decreased type 2 intermediate-stage progenitor cells in the hippocampal neurogenesis. Myelin vacuolation might be responsible for changes in neurogenesis possibly by reducing nerve conduction velocity of cholinergic inputs from the septal-hippocampal pathway to granule cell lineages and/or GABAergic interneurons, and of glutamatergic inputs to granule cell lineages.

  1. Mechanism of Cytosolic Phospholipase A2 Activation in Ghrelin Protection of Salivary Gland Acinar Cells against Ethanol Cytotoxicity

    Directory of Open Access Journals (Sweden)

    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Ghrelin, a peptide hormone, newly identified in oral mucosal tissues, has emerged recently as an important mediator of the processes of mucosal defense. Here, we report on the mechanism of ghrelin protection against ethanol cytotoxicity in rat sublingual salivary gland cells. The protective effect of ghrelin was associated with the increase in NO and PGE2, and upregulation in cytosolic phospholipase A2 (cPLA2 activity and arachidonic acid (AA release. The loss in countering effect of ghrelin occurred with cNOS inhibitor, L-NAME, as well as indomethacin and COX-1 inhibitor, SC-560, while COX-2 inhibitor, NS-398, and iNOS inhibitor, 1400W, had no effect. The effect of L-NAME was reflected in the inhibition of ghrelin-induced cell capacity for NO production, cPLA2 activation and PGE2 generation, whereas indomethacin caused only the inhibition in PGE2. Moreover, the ghrelin-induced up-regulation in AA release was reflected in the cPLA2 phosphorylation and S-nitrosylation. Inhibition in ghrelin-induced S-nitrosylation was attained with L-NAME, whereas the ERK inhibitor, PD98059, caused the blockage in cPLA2 protein phosphorylation as well as S-nitrosylation. Thus, ghrelin protection of salivary gland cells against ethanol involves cNOS-derived NO induction of cPLA2 activation through S-nitrosylation for the increase in AA release at the site of COX-1 action for PGE2 synthesis.

  2. Acinar-to-ductal metaplasia accompanies c-myc-induced exocrine pancreatic cancer progression in transgenic rodents.

    Science.gov (United States)

    Grippo, Paul J; Sandgren, Eric P

    2012-09-01

    Several important characteristics of exocrine pancreatic tumor pathogenesis remain incompletely defined, including identification of the cell of origin. Most human pancreatic neoplasms are ductal adenocarcinomas. However, acinar cells have been proposed as the source of some ductal neoplasms through a process of acinar-to-ductal metaplasia. The oncogenic transcription factor c-myc is associated with human pancreatic neoplasms. Transgenic mice overexpressing c-myc under control of acinar cell-specific elastase (Ela) gene regulatory elements not only develop acinar cell carcinomas but also mixed neoplasms that display both acinar-like neoplastic cells and duct-like neoplastic cells. In this report, we demonstrate that, first, c-myc is sufficient to induce acinar hyperplasia, though neoplastic lesions develop focally. Second, cell proliferation remains elevated in the neoplastic duct cell compartment of mixed neoplasms. Third, the proliferation/apoptosis ratio in cells from all lesion types remains constant, suggesting that differential regulation of these processes is not a feature of cancer progression in this model. Fourth, before the development of mixed neoplasms, there is transcriptional activation of the duct cell-specific cytokeratin-19 gene promoter in multicellular foci of amylase-positive acinar neoplasms. This observation provides direct evidence for metaplasia as the mechanism underlying development of ductal neoplastic cells within the context of an acinar neoplasm and suggests that the stimulus for this transformation acts over a multicellular domain or field within a neoplasm. Finally, focal ductal elements develop in some acinar cell carcinomas in Ela-c-myc transgenic rats, indicating that myc-associated acinar-to-ductal metaplasia is not restricted to the mouse.

  3. Damage to pancreatic acinar cells and preservation of islets of Langerhans in a rat model of acute pancreatitis induced by Karwinskia humboldtiana (buckthorn).

    Science.gov (United States)

    Carcano-Diaz, Katya; Garcia-Garcia, Aracely; Segoviano-Ramirez, Juan Carlos; Rodriguez-Rocha, Humberto; Loera-Arias, Maria de Jesus; Garcia-Juarez, Jaime

    2016-09-01

    Karwinskia humboldtiana (Kh) is a poisonous plant that grows in some regions of the American continent. Consuming large amounts of Kh fruit results in acute intoxication leading to respiratory failure, culminating in death within days. There is evidence of histological damage to the lungs, liver, and kidneys following accidental and experimental Kh intoxication. To date, the microscopic effect of Kh consumption on the pancreas has not been described. We examined the early effects of Kh fruit on pancreatic tissue at different stages of acute intoxication in the Wistar rat. We found progressive damage confined to the exocrine pancreas, starting with a reduction in the number of zymogen granules, loss of acinar architecture, the presence of autophagy-like vesicles, apoptosis and inflammatory infiltrate. The pancreatic pathology culminated in damaged acini characterized by necrosis and edema, with a complete loss of lobular architecture. Interestingly, the morphology of the islets of Langerhans was conserved throughout our evaluations. Taken together, our results indicate the damage induced by a high dose of Kh fruit in the Wistar rat is consistent with an early acute necrotizing pancreatitis that exclusively affects the exocrine pancreas. Therefore, this system might be useful as an animal model to study the treatment of pancreatic diseases. More importantly, as the islets of Langerhans were preserved, the active compounds of Kh fruit could be utilized for the treatment of acinar pancreatic cancer. Further studies might provide insight into the severity of acute Kh intoxication in humans and influence the design of treatments for pancreatic diseases and acinar pancreatic cancer. PMID:26877198

  4. Clinicopathologic analyses of salivary gland acinar cell carcinoma and review of the literature%涎腺腺泡细胞癌临床病理分析并文献复习

    Institute of Scientific and Technical Information of China (English)

    刘畅; 许春伟; 王晶晶; 张立英

    2016-01-01

    Objective: To investigate the clinicopathological features and diagnostic criteria of salivary gland acinar cell carcinoma.Methods:Retrospectively analyzed the features of clinicopathology and immunohistochemistry of a case of salivary gland acinar cell carcinoma, and combined together the review of the literatures.Results: Under light microscope, the tumor cell body was wide, and basophilic cytoplasm was ifne granular, nuclear round tumor cell growth was gland bubbly or slice solid growth, area of tumor was the papillary change, this case showed positive immunostaining for AE1/AE3, CK8/18, CK7, AAT and S-100.Conclusion: Salivary gland acinar cell carcinoma has a low incidence, but its frequent position of invasion and typical histology shape, combining together immunohistochemistry methods, will make for diagnosis and differential diagnosis possible.%目的:探讨涎腺腺泡细胞癌的临床病理学特点及诊断要点。方法:对1例涎腺腺泡细胞癌进行临床资料、病理形态学及免疫组织化学观察,并结合文献对其诊断及鉴别诊断进行探讨。结果:镜下瘤细胞胞体宽大,胞浆嗜碱性呈细颗粒状,核圆形瘤细胞生长呈腺泡状或实性片状生长,部分区域呈乳头状改变,免疫组化显示AE1/AE3(+)、CK8/18(+)、CK7(+)、AAT(+)、S-100(+)。结论:涎腺腺泡细胞癌发病率低,但根据其常见的发病部位及特征性的组织形态,结合免疫组织化学方法,有助于其诊断及鉴别诊断。

  5. H1-antihistamines induce vacuolation in astrocytes through macroautophagy.

    Science.gov (United States)

    Hu, Wei-Wei; Yang, Ying; Wang, Zhe; Shen, Zhe; Zhang, Xiang-Nan; Wang, Guang-Hui; Chen, Zhong

    2012-04-15

    H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine>pyrilamine>astemizole>triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5⁻/⁻ mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the major

  6. H1-antihistamines induce vacuolation in astrocytes through macroautophagy

    International Nuclear Information System (INIS)

    H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5−/− mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the major

  7. H1-antihistamines induce vacuolation in astrocytes through macroautophagy

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wei-Wei; Yang, Ying; Wang, Zhe; Shen, Zhe; Zhang, Xiang-Nan [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China); Wang, Guang-Hui [College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123 (China); Chen, Zhong, E-mail: chenzhong@zju.edu.cn [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China)

    2012-04-15

    H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5{sup −/−} mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the

  8. Vacuolation induced by unfavorable pH in cyanobacteria

    Institute of Scientific and Technical Information of China (English)

    赵以军; 吴红艳; 郭厚良; 许敏; 程凯; 祝海燕

    2001-01-01

    Six species or strains of cyanobacteria, Anabaena sp. 595, Plectonema boryanum 246, Scytonema hofmanni 248, Nostoc sp. 96, Oscillatoria animlis 284 and Spirulina maxima 438, were cultured in unfavorable pH conditions for vacuole induction. At pH 5.0, 6.5, or 7.0, vacuoles were observed to form in both Anabaena sp. 595 and Plectonema boryanum 246, especially in the former. The vacuolation took place with some morphological changes, such as the cells being inflated, spherical and vacuolated, and with unequalized division. The induced vacuoles in An- abaena sp. 595 and Plectonema boryanum 246 were in spherical shape and in rather transparent appearance under a phase microscope. For Scytonema hofmanni 248, it was less sensitive to pH, its vacuole formation was found only at pH 6.5. No vacuolization occurred in the cells of Nostoc sp. 96, Oscillatoria animlis 284 and Spirulina maxima 438 at all pH conditions we used. Vacuolization under unfavorable pH provides a new proof for the existence of vacuole in cells of cyanobacteria and reflects the prokaryote's function in ecological environment.

  9. FDG PET imaging of Ela1-myc mice reveals major biological differences between pancreatic acinar and ductal tumours

    Energy Technology Data Exchange (ETDEWEB)

    Abasolo, Ibane [Institut Municipal d' Investigacio Medica-Hospital del Mar, Parc de Recerca Biomedica de Barcelona, Barcelona (Spain); Universitat Pompeu Fabra, Parc de Recerca Biomedica de Barcelona, Departament de Ciencies Experimentals i de la Salut, Barcelona (Spain); Institut d' Alta Tecnologia - CRC, Parc de Recerca Biomedica de Barcelona, Barcelona (Spain); Pujal, Judit; Navarro, Pilar [Institut Municipal d' Investigacio Medica-Hospital del Mar, Parc de Recerca Biomedica de Barcelona, Barcelona (Spain); Rabanal, Rosa M.; Serafin, Anna [Universitat Autonoma de Barcelona, Departament de Medicina i Cirurgia Animals, Barcelona (Spain); Millan, Olga [Institut d' Alta Tecnologia - CRC, Parc de Recerca Biomedica de Barcelona, Barcelona (Spain); Real, Francisco X. [Institut Municipal d' Investigacio Medica-Hospital del Mar, Parc de Recerca Biomedica de Barcelona, Barcelona (Spain); Universitat Pompeu Fabra, Parc de Recerca Biomedica de Barcelona, Departament de Ciencies Experimentals i de la Salut, Barcelona (Spain); Programa de Patologia Molecular, Centro Nacional de Investigaciones Oncologicas, Madrid (Spain)

    2009-07-15

    The aim was to evaluate FDG PET imaging in Ela1-myc mice, a pancreatic cancer model resulting in the development of tumours with either acinar or mixed acinar-ductal phenotype. Transversal and longitudinal FDG PET studies were conducted; selected tissue samples were subjected to autoradiography and ex vivo organ counting. Glucose transporter and hexokinase mRNA expression was analysed by quantitative reverse transcription polymerase chain reaction (RT-PCR); Glut2 expression was analysed by immunohistochemistry. Transversal studies showed that mixed acinar-ductal tumours could be identified by FDG PET several weeks before they could be detected by hand palpation. Longitudinal studies revealed that ductal - but not acinar - tumours could be detected by FDG PET. Autoradiographic analysis confirmed that tumour areas with ductal differentiation incorporated more FDG than areas displaying acinar differentiation. Ex vivo radioactivity measurements showed that tumours of solely acinar phenotype incorporated more FDG than pancreata of non-transgenic littermates despite the fact that they did not yield positive PET images. To gain insight into the biological basis of the differential FDG uptake, glucose transporter and hexokinase transcript expression was studied in microdissected tumour areas enriched for acinar or ductal cells and validated using cell-specific markers. Glut2 and hexokinase I and II mRNA levels were up to 20-fold higher in ductal than in acinar tumours. Besides, Glut2 protein overexpression was found in ductal neoplastic cells but not in the surrounding stroma. In Ela1-myc mice, ductal tumours incorporate significantly more FDG than acinar tumours. This difference likely results from differential expression of Glut2 and hexokinases. These findings reveal previously unreported biological differences between acinar and ductal pancreatic tumours. (orig.)

  10. Regeneration of a lytic central vacuole and of neutral peripheral vacuoles can be visualized by green fluorescent proteins targeted to either type of vacuoles.

    Science.gov (United States)

    Di Sansebastiano, G P; Paris, N; Marc-Martin, S; Neuhaus, J M

    2001-05-01

    Protein trafficking to two different types of vacuoles was investigated in tobacco (Nicotiana tabacum cv SR1) mesophyll protoplasts using two different vacuolar green fluorescent proteins (GFPs). One GFP is targeted to a pH-neutral vacuole by the C-terminal vacuolar sorting determinant of tobacco chitinase A, whereas the other GFP is targeted to an acidic lytic vacuole by the N-terminal propeptide of barley aleurain, which contains a sequence-specific vacuolar sorting determinant. The trafficking and final accumulation in the central vacuole (CV) or in smaller peripheral vacuoles differed for the two reporter proteins, depending on the cell type. Within 2 d, evacuolated (mini-) protoplasts regenerate a large CV. Expression of the two vacuolar GFPs in miniprotoplasts indicated that the newly formed CV was a lytic vacuole, whereas neutral vacuoles always remained peripheral. Only later, once the regeneration of the CV was completed, the content of peripheral storage vacuoles could be seen to appear in the CV of a third of the cells, apparently by heterotypic fusion. PMID:11351072

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

    Science.gov (United States)

    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.

  12. Postmortem acinar autolysis in rat sublingual gland: a morphometric study

    Directory of Open Access Journals (Sweden)

    Leticia Rodrigues Nery

    2010-10-01

    Full Text Available ABSTRACT OBJECTIVE: To analyze and to quantify morphological acinar postmortem changes in rat sublingual glands (SLG. MATERIAL AND METHODSs: Fifty rats were divided into two groups of 25 animals each. Group I was used for morphological and morphometric evaluations and group II for the determination of gland density and processed gland volume. Acinar autolytic changes were studied at 0 (control group, 3, 6, 12 and 24 h postmortem periods. The morphometric analysis of the volume density (Vv and total volume (Vt of intact (ia and autolyzed (aa acini was performed under light microscopy using a Zeiss II integration grid with 100 symmetrically distributed points. RESULTS: Morphologically, temporal progressive nuclear alterations and gradual loss of the structural architecture of acinar cells were found. Regarding quantitative results, both the Vvaa and the Vvia showed statistically significant differences among all postmortem periods (p0.05, respectively. Vtaa increased from 0.18 mm³ at 0 h to 38.17 mm³ at 12 h, while Vtia showed a decrease from 33.47 mm³ to 0 mm³ between 3-24 h postmortem. Data concerning Vtaa were adjusted by two-variable linear regression, obtaining the equation: y=-3.54 + 3.38x (r²=0.90. The Vtaa growth rate calculated by this equation was 3.38 mm³/h between 0-12 h. CONCLUSION: Acinar autolysis on rat SLG demonstrated the most significant signs during the first 6 h postmortem and was widely spread through the gland at 12 h.

  13. Monoclonal Antibody 16D10 to the C-Terminal Domain of the Feto-Acinar Pancreatic Protein Binds to Membrane of Human Pancreatic Tumoral SOJ-6 Cells and Inhibits the Growth of Tumor Xenografts

    Directory of Open Access Journals (Sweden)

    Laurence Panicot-Dubois

    2004-11-01

    Full Text Available Feto-acinar pancreatic protein (FAPP characterized by mAbJ28 reactivity is a specific component associated with ontogenesis and behaves as an oncodevelopment-associated antigen. We attempted to determine whether pancreatic tumoral SOJ-6 cells are expressed at their surface FAPP antigens and to examine if specific antibodies directed against these FAPP epitopes could decrease the growth of pancreatic tumors in a mice model. For this purpose, we used specific antibodies against either the whole FAPP, the O-glycosylated C-terminal domain, or the N-terminal domain of the protein. Our results indicate that SOJ-6 cells expressed at their surface a 32-kDa peptide corresponding to the C-terminal domain of the FAPP. Furthermore, we show, by using endoproteinase Lys-C or geldanamycin, a drug able to impair the FAPP secretion, that this 32-kDa peptide expressed on the SOJ-6 cell surface comes from the degradation of the FAPP. Finally, an in vivo prospective study using a preventative tumor model in nude mice indicates that targeting this peptide by the use of mAb16D10 inhibits the growth of SOJ-6 xenografts. The specificity of mAb16D10 for pancreatic tumors and the possibility to obtain recombinant structures of mucin-like peptides recognized by mAb16D10 and mAbJ28 are promising tools in immunologic approaches to cure pancreatic cancers.

  14. 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.

    Science.gov (United States)

    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. PMID:25941231

  15. Different Degree in Proteasome Malfunction Has Various Effects on Root Growth Possibly through Preventing Cell Division and Promoting Autophagic Vacuolization

    OpenAIRE

    Xianyong Sheng; Qian Wei; Liping Jiang; Xue Li; Yuan Gao; Li Wang

    2012-01-01

    The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4-64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated protei...

  16. Different degree in proteasome malfunction has various effects on root growth possibly through preventing cell division and promoting autophagic vacuolization.

    Directory of Open Access Journals (Sweden)

    Xianyong Sheng

    Full Text Available The ubiquitin/proteasome pathway plays a vital role in plant development. But the effects of proteasome malfunction on root growth, and the mechanism underlying this involvement remains unclear. In the present study, the effects of proteasome inhibitors on Arabidopsis root growth were studied through the analysis of the root length, and meristem size and cell length in maturation zone using FM4-64, and cell-division potential using GFP fusion cyclin B, and accumulation of ubiquitinated proteins using immunofluorescence labeling, and autophagy activity using LysoTracker and MDC. The results indicated that lower concentration of proteasome inhibitors promoted root growth, whereas higher concentration of inhibitors had the opposite effects. The accumulation of cyclin B was linked to MG132-induced decline in meristem size, indicating that proteasome malfunction prevented cell division. Besides, MG132-induced accumulation of the ubiquitinated proteins was associated with the increasing fluorescence signal of LysoTracker and MDC in the elongation zone, revealing a link between the activation of autophagy and proteasome malfunction. These results suggest that weak proteasome malfunction activates moderate autophagy and promotes cell elongation, which compensates the inhibitor-induced reduction of cell division, resulting in long roots. Whereas strong proteasome malfunction induces severe autophagy and disturbs cell elongation, resulting in short roots.

  17. Root bark extracts of Juncus effusus and Paeonia suffruticosa protect salivary gland acinar cells from apoptotic cell death induced by cis-platinum (II) diammine dichloride.

    Science.gov (United States)

    Mukudai, Yoshiki; Kondo, Seiji; Shiogama, Sunao; Koyama, Tomoyuki; Li, Chunnan; Yazawa, Kazunaga; Shintani, Satoru

    2013-12-01

    Cis-platinum (II) diammine dichloride (CDDP) is a platinum-based anticancer agent, and is often used for chemotherapy for malignant tumors, albeit CDDP has serious side-effects, including xerostomia (dry mouth). Since patients with xerostomia have reduced quality of life, it is urgent and important to identify nontoxic and natural agents capable of reducing the adverse effect of chemotherapy on salivary gland function. Therefore, we commenced an institutional collaborative project in which candidates of herbal extracts were selected from more than 400 bioactive herbal products for their potential therapeutic effects not only on xerostomia, but also on oral diseases. In the present study, we report on two Chinese medical herbal extracts from the root barks of Juncus effusus and Paeonia suffruticosa. The two extracts showed a protective effect in NS-SV-Ac cells from the cytotoxicity and apoptosis caused by CDDP. The effect was dependent on the p53 pathway, protein kinase B/Akt 1 and mitochondrial apoptosis-related proteins (i.e. Bcl-2 and Bax), but was not dependent on nuclear factor κB. Notably, the apoptosis-protective effect of the extracts was not observed in adenocystic carcinoma cell lines. Although these extracts have been utilized in traditional Chinese medicine for hundreds of years, there are no reports to our knowledge, on their therapeutic effects on xerostomia. Thus, in the present study, we elucidated the potency of these herbal extracts as novel candidates for xerostomia to improve the quality of life of patients undergoing chemotherapy.

  18. Helicobacter pylori vacuolating toxin A and apoptosis

    Directory of Open Access Journals (Sweden)

    Rassow Joachim

    2011-11-01

    Full Text Available Abstract VacA, the vacuolating cytotoxin A of Helicobacter pylori, induces apoptosis in epithelial cells of the gastic mucosa and in leukocytes. VacA is released by the bacteria as a protein of 88 kDa. At the outer surface of host cells, it binds to the sphingomyelin of lipid rafts. At least partially, binding to the cells is facilitated by different receptor proteins. VacA is internalized by a clathrin-independent mechanism and initially accumulates in GPI-anchored proteins-enriched early endosomal compartments. Together with early endosomes, VacA is distributed inside the cells. Most of the VacA is eventually contained in the membranes of vacuoles. VacA assembles in hexameric oligomers forming an anion channel of low conductivity with a preference for chloride ions. In parallel, a significant fraction of VacA can be transferred from endosomes to mitochondria in a process involving direct endosome-mitochondria juxtaposition. Inside the mitochondria, VacA accumulates in the mitochondrial inner membrane, probably forming similar chloride channels as observed in the vacuoles. Import into mitochondria is mediated by the hydrophobic N-terminus of VacA. Apoptosis is triggered by loss of the mitochondrial membrane potential, recruitment of Bax and Bak, and release of cytochrome c.

  19. cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in aleurone cells and inhibits GA-dependent hydrolysis of storage protein and phytate.

    Science.gov (United States)

    Hwang, Yong-sic; Bethke, Paul C; Gubler, Frank; Jones, Russell L

    2003-07-01

    The putative H+/Cl- symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when aleurone cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in aleurone layers and prevents synthesis of secretory proteins such as alpha-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the aleurone layer. Vacuolation of the aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl- symporter in aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of alpha-amylase when Cl- ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated cells.

  20. Calcium Signals from the Vacuole

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    Gerald Schönknecht

    2013-10-01

    Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

  1. Magnesium Sensitizes Slow Vacuolar Channels to Physiological Cytosolic Calcium and Inhibits Fast Vacuolar Channels in Fava Bean Guard Cell Vacuoles.

    Science.gov (United States)

    Pei; Ward; Schroeder

    1999-11-01

    Vacuolar ion channels in guard cells play important roles during stomatal movement and are regulated by many factors including Ca(2+), calmodulin, protein kinases, and phosphatases. We report that physiological cytosolic and luminal Mg(2+) levels strongly regulate vacuolar ion channels in fava bean (Vicia faba) guard cells. Luminal Mg(2+) inhibited fast vacuolar (FV) currents with a K(i) of approximately 0.23 mM in a voltage-dependent manner at positive potentials on the cytoplasmic side. Cytosolic Mg(2+) at 1 mM also inhibited FV currents. Furthermore, in the absence of cytosolic Mg(2+), cytosolic Ca(2+) at less than 10 µM did not activate slow vacuolar (SV) currents. However, when cytosolic Mg(2+) was present, submicromolar concentrations of cytosolic Ca(2+) activated SV currents with a K(d) of approximately 227 nM, suggesting a synergistic Mg(2+)-Ca(2+) effect. The activation potential of SV currents was shifted toward physiological potentials in the presence of cytosolic Mg(2+) concentrations. The direction of SV currents could also be changed from outward to both outward and inward currents. Our data predict a model for SV channel regulation, including a cytosolic binding site for Ca(2+) with an affinity in the submicromolar range and a cytosolic low-affinity Mg(2+)-Ca(2+) binding site. SV channels are predicted to contain a third binding site on the vacuolar luminal side, which binds Ca(2+) and is inhibitory. In conclusion, cytosolic Mg(2+) sensitizes SV channels to physiological cytosolic Ca(2+) elevations. Furthermore, we propose that cytosolic and vacuolar Mg(2+) concentrations ensure that FV channels do not function as a continuous vacuolar K(+) leak, which would prohibit stomatal opening. PMID:10557247

  2. Research of TAP Induction The Pancreatic Acinar Cells to Release HMGB1 in Rat%TAP诱导大鼠胰腺腺泡细胞释放HMGB1的研究

    Institute of Scientific and Technical Information of China (English)

    王国良; 兑丹华; 白亮; 刘尧; 田飞; 魏巍

    2012-01-01

    目的 探讨人胰蛋白酶原激活肽(TAP)诱导大鼠胰腺腺泡细胞高迁移率族蛋白1(HMGB1)的分泌规律和丙酮酸乙酯(EP)对其释放的影响.方法 将12只SD大鼠处死后,取出胰腺分离胰腺腺泡细胞,将所得细胞悬液按抽签法随机分为3组,对照组、TAP组及EP组.TAP组及EP组加入相同剂量的TAP(终浓度3 nmol/L),EP组同时加入EP(终浓度28 mmol/L),在3、6、12及24 h分别取细胞样本.采用实时定量逆转录聚合酶链反应(RT-PCR)检测HMGB1 mRNA的表达,蛋白印迹法(Western blot)检测HMGB1蛋白的表达;并进行HMGB1 mRNA及蛋白的表达与TAP作用时间的Spearman秩相关分析.结果 与对照组比较,TAP组及EP组的HMGB1 mRNA及蛋白的表达均随TAP作用时间的延长而上调(P<0.05);与TAP组比较,EP组的HMGB1mRNA及蛋白表达则下调(P<0.05).TAP组组内比较显示,HMGB1 mRNA及蛋白的表达随TAP作用时间延长而逐渐升高(P<0.05),且以12h及24 h时升高明显(P<0.01),HMGB1 mRNA及蛋白的表达与TAP作用时间呈正相关(rs=0.971,P<0.01;rs=0.966,P<0.01).结论 TAP可诱导大鼠胰腺腺泡细胞内HMGB1的释放.急性胰腺炎早期的TAP与晚期的HMGB1之间存在着时间的正向联系.EP可抑制HMGB1的释放.%Objective To explore the secretion law of high mobility group box 1 (HMGB1) in rat pancreatic acinar cells induced by trypsin activation peptide (TAP) and release of HMGB1 affected by ethyl pyruvate (EP) . Methods The experiment was performed in 12 SD rats. The pancreatic acinar cells of rats were taken out and then separated into three groups: control group, TAP group, and EP group. TAP was added into TAP group and EP group (keep TAP at a final concentration of 3 nmol/L), respectively, but EP was added into EP group only (keep EP at a final concentration of 28 mmol/L). The expressions of HMGB1 mRNA and protein were detected by using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) or

  3. Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study

    Directory of Open Access Journals (Sweden)

    Luciana Reis AZEVEDO-ALANIS

    2015-10-01

    Full Text Available Although some morphological investigations on aged human sublingual glands (HSG found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated.Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death.Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years; II (31–60, and III (61–90. Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM. Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05.Results There was correlation between age and acinar autolysis (r=0.38; p=0.0001. However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis.Conclusion Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death.

  4. Acinar autolysis and mucous extravasation in human sublingual glands: a microscopic postmortem study

    Science.gov (United States)

    AZEVEDO-ALANIS, Luciana Reis; TOLENTINO, Elen de Souza; de ASSIS, Gerson Francisco; CESTARI, Tânia Mary; LARA, Vanessa Soares; DAMANTE, José Humberto

    2015-01-01

    Although some morphological investigations on aged human sublingual glands (HSG) found eventual phenomena identified as autolysis and mucous extravasation, the exact meaning of these findings has not been elucidated. Objective The aim of this work is to investigate whether acinar autolysis and mucous extravasation are related to the aging process in human sublingual glands. We also speculate if autolytic changes may assist forensic pathologists in determining time of death. Material and Methods 186 cadavers’ glands were allocated to age groups: I (0–30 years); II (31–60), and III (61–90). Time and mode of death were also recorded. Acinar autolysis and mucous extravasation were classified as present or absent. Ultrastructural analysis was performed using transmission electron microscopy (TEM). Data were compared using Mann-Whitney U, Spearman’s correlation coefficient, Kruskal-Wallis, and Dunn tests (p<0.05). Results There was correlation between age and acinar autolysis (r=0.38; p=0.0001). However, there was no correlation between autolysis and time of death. No differences were observed between genders. TEM showed mucous and serous cells presenting nuclear and membrane alterations and mucous cells were more susceptible to autolysis. Conclusion Acinar autolysis occurred in all age groups and increased with age while mucous extravasation was rarely found. Both findings are independent. Autolysis degrees in HSG could not be used to determine time of death. PMID:26537715

  5. Reactive oxygen species (ROS) is not a promotor of taxol-induced cytoplasmic vacuolization

    Science.gov (United States)

    Sun, Qingrui; Chen, Tongsheng

    2009-02-01

    we have previously reported that taxol, a potent anticancer agent, induces caspase-independent cell death and cytoplasmic vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. However, the mechanisms of taxol-induced cytoplasmic vacuolization are poorly understood. Reactive oxygen species (ROS) has been reported to be involved in the taxol-induced cell death. Here, we employed confocal fluorescence microscopy imaging to explore the role of ROS in taxol-induced cytoplasmic vacuolization. We found that ROS inhibition by addition of N-acetycysteine (NAC), a total ROS scavenger, did not suppress these vacuolization but instead increased vacuolization. Take together, our results showed that ROS is not a promotor of the taxol-induced cytoplasmic vacuolization.

  6. p21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

    Science.gov (United States)

    Grabliauskaite, Kamile; Hehl, Adrian B; Seleznik, Gitta M; Saponara, Enrica; Schlesinger, Kathryn; Zuellig, Richard A; Dittmann, Anja; Bain, Martha; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-02-01

    Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. PMID:25212177

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

    Science.gov (United States)

    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

  8. Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments.

    Science.gov (United States)

    Papini, E; de Bernard, M; Milia, E; Bugnoli, M; Zerial, M; Rappuoli, R; Montecucco, C

    1994-01-01

    Pathogenic strains of Helicobacter pylori cause progressive vacuolation and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, transferrin, and transferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by affinity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findings indicate that the vacuoles specifically originate from late endosomal compartments. Images PMID:7937879

  9. Induction of C-FOS, C-MYC and P53 by US -adrenergic receptor (US -AR) stimulation of rat parotid acinar cells (RPAC)

    Energy Technology Data Exchange (ETDEWEB)

    Kousvelari, E.E.; Louis, J.; Curran, T.; Baum, B.J.

    1987-05-01

    Treatment of rats with the US -agonist isoproterenol (ISO) results in dramatically increased parotid gland protein synthesis, processing and cell proliferation. The authors have shown that in RPAC in vitro, US -AR stimulation has similar effect on protein synthesis and processing. Proto-oncogenes have been implicated in growth regulation, differentiation and in mediating some extracellular stimulated events at the level of gene expression. To understand the regulation of cellular events after US -AR stimulation, the expression of c-fos, c-myc and p53 was investigated. RPAC were incubated with or without 10 VM ISO for 15, 30, 60 min. mRNA was isolated from cells and hybridization analysis was performed on nitrocellulose paper-transferred mRNA using TSP-labeled DNA probes. At early time points, the levels of c-fos gene activation in ISO-treated and control cells were comparable. After 60 min of ISO treatment, a sharp 20-30 fold induction of c-fos expression occurred. Similar increases in c-myc and p53 gene expression were observed after 60 min of ISO treatment. The authors data indicate that early effects of US -AR stimulation of RPAC include induction of c-fos, c-myc and p53 gene expression as well as enhanced protein synthesis and processing.

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

    Science.gov (United States)

    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. PMID:21611741

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

    Science.gov (United States)

    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.

  12. Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices

    Directory of Open Access Journals (Sweden)

    Goldenberg Anna

    2006-06-01

    Full Text Available Abstract Background Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries. Methods Intra-acinar arteries of the mouse lung were studied in 200 μm thick precision-cut lung slices (PCLS. The organisation of the muscle coat of these vessels was characterized by α-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed. Results Intra-acinar arteries are equipped with a discontinuous spiral of α-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2 that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone and III (antimycin A specifically interfered with HPV, whereas blockade of complex IV (sodium azide unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction. Conclusion This study establishes the first model for investigation of basic characteristics of HPV

  13. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    Science.gov (United States)

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  14. Vps1 in the late endosome-to-vacuole traffic.

    Science.gov (United States)

    Hayden, Jacob; Williams, Michelle; Granich, Ann; Ahn, Hyoeun; Tenay, Brandon; Lukehart, Joshua; Highfill, Chad; Dobard, Sarah; Kim, Kyoungtae

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 delta cells accumulated FM4-64 to a greater extent than wild-type (WT))cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole. PMID:23385815

  15. Vps1 in the late endosome-to-vacuole traffic

    Indian Academy of Sciences (India)

    Jacob Hayden; Michelle Williams; Ann Granich; Hyoeun Ahn; Brandon Tenay; Joshua Lukehart; Chad Highfill; Sarah Dobard; Kyoungtae Kim

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.

  16. Nitric Oxide Inhibits Coxiella burnetii Replication and Parasitophorous Vacuole Maturation

    Science.gov (United States)

    Howe, Dale; Barrows, Lorraine F.; Lindstrom, Nicole M.; Heinzen, Robert A.

    2002-01-01

    Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellular Coxiella burnetii in murine L-929 cells. Immunoblotting and nitrite assays revealed that C. burnetii infection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitor S-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect on C. burnetii replication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic of C. burnetii infection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing one C. burnetii cell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multiple C. burnetii cells. These experiments demonstrate that nitric oxide reversibly inhibits replication of C. burnetii and formation of the parasitophorous vacuole. PMID:12183564

  17. 姜黄素对长期摄入酒精和不同量蛋白质的大鼠胰腺腺泡细胞损伤的保护作用研究%Effects of Curcumin on Pancreatic Acinar Cell Injury in Rats with Long-term Alcohol Intake and Different Amount of Protein

    Institute of Scientific and Technical Information of China (English)

    周旭春

    2011-01-01

    目的:研究姜黄素对长期摄入酒精和不同量蛋白质的大鼠胰腺腺泡细胞损伤的保护作用.方法:实验分为5组,即正常对照(正常饲养)、高蛋白、低蛋白、高蛋白+姜黄素、低蛋白+姜黄素(以25%酒精代替饮水自由饮用,高、低蛋白质占总热量供给的32%、6%,喂饲6个月)组.在光镜和电镜下观察大鼠胰腺腺泡细胞结构变化,用比色法检测胰腺组织匀浆淀粉酶和脂肪酶的含量,TUNEL法检测腺泡细胞凋亡情况,免疫组化检测胰腺组织切片中环氧化酶-2(COX-2)的变化.结果:与高、低蛋白组比较,高、低蛋白+姜黄素组大鼠胰腺腺泡细胞髓样结构减少,线粒体肿胀减轻;淀粉酶和脂肪酶含量均显著升高(P<0.05);胰腺腺泡细胞凋亡显著减少(P<0.05);COX-2的表达降低.结论:姜黄素可预防摄入酒精联合过高或过低蛋白质的大鼠胰腺腺泡细胞损伤,延缓酒精性胰腺损伤的进程.%OBJECTIVE: To investigate the protective effects of curcumin on pancreatic acinar cell injury in rats with long-term alcohol intake and protein consumption. METHODS: Wistar rats were divided into 5 groups, I.e. Normal control group (the group fed with normal feed) ,high protein group, low protein group, high protein+curcumin group, low protein+curcumin group (those groups fed with diet containing 25% ethanol instead of drinking water for 6 months). High and low protein accounted for 32% and 6 % of total heat quantity. The structure change of pancreatic acinar cell was observed under light microscope and electron microscope. The contents of amylase and lipase in pancreatic tissue homogenate were determined by colorimetry. Apoptosis and expression of cyclooxygenase-2(COX-2) in acinar cell were detected by TUNEL and immunohistochemical staining, respectively. RESULTS: Compared with no application of curcumin, myelin figure and enlarged mitochondria were reduced in curcumin treatment groups. Lipase and amylase

  18. Endothelial tubes assemble from intracellular vacuoles in vivo.

    Science.gov (United States)

    Kamei, Makoto; Saunders, W Brian; Bayless, Kayla J; Dye, Louis; Davis, George E; Weinstein, Brant M

    2006-07-27

    The formation of epithelial tubes is crucial for the proper development of many different tissues and organs, and occurs by means of a variety of different mechanisms. Morphogenesis of seamless, properly patterned endothelial tubes is essential for the development of a functional vertebrate circulatory system, but the mechanism of vascular lumenization in vivo remains unclear. Evidence dating back more than 100 years has hinted at an important function for endothelial vacuoles in lumen formation. More than 25 years ago, in some of the first endothelial cell culture experiments in vitro, Folkman and Haudenschild described "longitudinal vacuoles" that "appeared to be extruded and connected from one cell to the next", observations confirmed and extended by later studies in vitro showing that intracellular vacuoles arise from integrin-dependent and cdc42/Rac1-dependent pinocytic events downstream of integrin-extracellular-matrix signalling interactions. Despite compelling data supporting a model for the assembly of endothelial tubes in vitro through the formation and fusion of vacuoles, conclusive evidence in vivo has been lacking, primarily because of difficulties associated with imaging the dynamics of subcellular endothelial vacuoles deep within living animals. Here we use high-resolution time-lapse two-photon imaging of transgenic zebrafish to examine how endothelial tubes assemble in vivo, comparing our results with time-lapse imaging of human endothelial-cell tube formation in three-dimensional collagen matrices in vitro. Our results provide strong support for a model in which the formation and intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation. PMID:16799567

  19. KLF4 Is Essential for Induction of Cellular Identity Change and Acinar-to-Ductal Reprogramming during Early Pancreatic Carcinogenesis.

    Science.gov (United States)

    Wei, Daoyan; Wang, Liang; Yan, Yongmin; Jia, Zhiliang; Gagea, Mihai; Li, Zhiwei; Zuo, Xiangsheng; Kong, Xiangyu; Huang, Suyun; Xie, Keping

    2016-03-14

    Understanding the molecular mechanisms of tumor initiation has significant impact on early cancer detection and intervention. To define the role of KLF4 in pancreatic ductal adenocarcinoma (PDA) initiation, we used molecular biological analyses and mouse models of klf4 gain- and loss-of-function and mutant Kras. KLF4 is upregulated in and required for acinar-to-ductal metaplasia. Klf4 ablation drastically attenuates the formation of pancreatic intraepithelial neoplasia induced by mutant Kras(G12D), whereas upregulation of KLF4 does the opposite. Mutant KRAS and cellular injuries induce KLF4 expression, and ectopic expression of KLF4 in acinar cells reduces acinar lineage- and induces ductal lineage-related marker expression. These results demonstrate that KLF4 induces ductal identity in PanIN initiation and may be a potential target for prevention of PDA initiation.

  20. A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways.

    Science.gov (United States)

    Fishler, Rami; Sznitman, Josué

    2016-01-01

    Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (µPIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between µPIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs. PMID:27214269

  1. Epiplakin deficiency aggravates murine caerulein-induced acute pancreatitis and favors the formation of acinar keratin granules.

    Directory of Open Access Journals (Sweden)

    Karl L Wögenstein

    Full Text Available Epiplakin, a member of the plakin protein family, is exclusively expressed in epithelial tissues and was shown to bind to keratins. Epiplakin-deficient (EPPK-/- mice showed no obvious spontaneous phenotype, however, EPPK-/- keratinocytes displayed faster keratin network breakdown in response to stress. The role of epiplakin in pancreas, a tissue with abundant keratin expression, was not yet known. We analyzed epiplakin's expression in healthy and inflamed pancreatic tissue and compared wild-type and EPPK-/- mice during caerulein-induced acute pancreatitis. We found that epiplakin was expressed primarily in ductal cells of the pancreas and colocalized with apicolateral keratin bundles in murine pancreatic acinar cells. Epiplakin's diffuse subcellular localization in keratin filament-free acini of K8-deficient mice indicated that its filament-associated localization in acinar cells completely depends on its binding partner keratin. During acute pancreatitis, epiplakin was upregulated in acinar cells and its redistribution closely paralleled keratin reorganization. EPPK-/- mice suffered from aggravated pancreatitis but showed no obvious regeneration phenotype. At the most severe stage of the disease, EPPK-/- acinar cells displayed more keratin aggregates than those of wild-type mice. Our data propose epiplakin to be a protective protein during acute pancreatitis, and that its loss causes impaired disease-associated keratin reorganization.

  2. The research of rat bone marrow mesenchymal stem cells differentiation into salivary gland acinar-like cells%诱导大鼠骨髓间充质干细胞分化为唾液腺腺泡样细胞的实验研究

    Institute of Scientific and Technical Information of China (English)

    梁亮; 孙沫逸; 李建虎; 杨永勤; 申志远; 苏忠平

    2012-01-01

    Objective To explore the possibility of rat bone marrow mesenchymal stem cells(MSCs) differentiation intosalivary gland acinar-like cells induced by co-culture with salivary gland acinar cells (SGCS) in vitro. Methods SGCsof passage 1 and MSCs of passage 3 were taken as subject investigated.Following groups were allocated:co-culturewith salivary gland medium group;co-culture with 10% FBS DMEM/F12 group;salivary gland medium group;group with10% FBS DMEM/F12.MSCs of every group were identified by immunohistochemiscal analysis withα -amylase After amonth cultivation.MSCs conversion rate is attained by calculation of the number of positive cells.By the opticalmicroscope and scanning electron microscope observes the morphological changes of cells. Results Compared withnone-co-cultured groups,Most cells in co-culture groups were stained by α-amylase(P<0.05). Conclusions The cellsof success induction morphology which similar to salivary gland acinar cells at the optical microscope and scanningelectron microscope display.MSCs could differentiate into SGs at phenotype and molecule level by co-culture of SGs invitro.Changing the experimental conditions and can improve induction efficiency.%目的:探讨在共培养系统下大鼠骨髓间充质干细胞分化为唾液腺腺泡样细胞的实验.方法:以纯化1代SD大鼠颌下腺腺泡细胞和3代骨髓间充质干细胞作为共培养实验对象.实验分组:含唾液腺培养液的共培养组;含10%FBS、DMEM/F12的共培养组;含唾液腺培养液的非共培养组;含10%FBS、DMEM/F12的非共培养组.培养1个月经α-淀粉酶(α-amylase)免疫组化染色各组的MSCs,计算阳性细胞数得出MSCs的转化率,并且通过光镜和电镜鉴定细胞形态变化.结果:各组诱导的MSCs经α-amylase染色,共培养组阳性细胞数较非共培养组多(P<0.05),且诱导成功的细胞在镜下形态类似于唾液腺腺泡细胞.结论:在共培养条件下成功实现了MSCs向 SGCs的形态学转

  3. Inhibition of proliferation by PERK regulates mammary acinar morphogenesis and tumor formation.

    Directory of Open Access Journals (Sweden)

    Sharon J Sequeira

    Full Text Available Endoplasmic reticulum (ER stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2alpha. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2alpha and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-DeltaC or PERK-K618A, does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKDeltaC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer.

  4. PTD-NBD polypeptide down-regulates expression of NF-κB p65 in inflammatory pancreatic acinar cell injury in rats%PTD-NBD多肽对大鼠胰腺腺泡细胞炎症损伤中NF-κB表达的影响

    Institute of Scientific and Technical Information of China (English)

    谢文瑞; 杨元生; 杨新魁; 陈垦; 陈婧华; 崔淑兰; 王晖

    2013-01-01

    To examine the effect of PTD-NBD polypeptide on the expression of nuclear factor κB (NF-κB) p65 in inflammatory pancreatic acinar cell injury in rats.METHODS:Rat pancreatic acinar cells were isolated,cultured,and divided into a normal control group,an acute pancreatitis (AP) group and a PTD-NBD polypeptides group.An in vitro model of AP was induced by treating rat pancreatic acinar cells with lipopolysaccharide (10 mg/L).Cell morphological changes were observed,and the contents of amylase,superoxide dismutase (SOD) and IL-1β in culture medium were tested.Expression of NF-κB p65 mRNA and protein in cells was detected by RT-PCR and Western blot 6 and 12 h after modeling,respectively.RESULTS:Compared to the control group,pancreatic acinar cell swelling and death were increased (6 h:8.9 ± 0.34 vs 1.1 ± 0.13; 12 h:9.4 ± 0.26 vs 1.2 ± 0.15,both P < 0.05),the contents of amylase (6 h:2135.8 ± 347.2 vs 873.5 ± 91.6; 12 h:3299.6 ± 217.7 vs 917.7 ± 101.9,both P < 0.05) and IL-1β (6 h:84.9 ± 15.7 vs 39.3 ± 7.9; 12 h:95.6 ± 17.1 vs 38.9 ± 5.2,both P < 0.05) were increased and the contents of SOD were decreased in culture medium (6 h:116.3 ± 30.3 vs 176.2 ± 21.6; 12h:101.5 ± 25.6 vs 173.6 ± 27.9,P < 0.05),and the expression of NF-kB p65 in pancreatic acinar ceils was increased (P < 0.05) in the AP group at 6 and 12 h after modeling.Compared to the AP group,pancreatic acinar cell swelling and death were lessened (6 h:6.8 ± 0.23 vs 8.9 ± 0.34; 12 h:7.5 ± 0.19 vs 9.4 ± 0.26,both P < 0.05),the contents of SOD were raised (6 h:137.6 ± 27.4 vs 116.3 ± 30.3; 12 h:144.3 ± 23.6 vs 101.5 ± 25.6,both P < 0.05)and the contents of amylase (6 h:1951.5 ± 211.7 vs 2135.8 ± 347.2; 12 h:1761.3 ± 231.5 vs 3299.6 ± 217.7,both P < 0.05) and IL-1β (6 h:66.8 ± 11.6 vs 84.9 ± 15.7; 12 h:54.8 ± 21.2 vs 95.6 ± 17.1,both P < 0.05) were decreased in culture medium,and the expression of NF-κB p65 mRNA and protein was down-regulated in the PAT

  5. Effect of the Vacuolation of Helicobacter Pylori

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cytotoxic test in vitro combined with cytochemical stain, fluorescent stain, transmission electronmicrograph was used to study the vacuolated effect by helicobacter pylori (H.pylori) (Toxin+) and its pathological mechanism. 78.26 % patients with peptic ulcer associated with H.pylori was infected with H.pylori (Toxin+), while 42.86 % patients with gastritis was infected with H.pylori (Toxin+). It was positive in vacuole with acridine orange and acid phosphatase stain. Transmission electronmicrograph of vacuole revealed the presence of abounding membrane. There was a closed relationship between infection with H.pylori (Toxin+) and peptic ulcer disease. The vacuole induced by H.pylori (Toxin+) was autophagosome, which was pathological phenomenon induced by toxin.

  6. Cytotoxic effect of desoxycholic acid on pancreatic acinar cells and its influence on the activity of nuclear transcription factors%脱氧胆酸对胰腺腺泡细胞的损伤及核转录因子活性的影响

    Institute of Scientific and Technical Information of China (English)

    张桂信; 陈海龙; 曹传海; 林小洋; 张利; 纪军; 王永鹏

    2011-01-01

    目的 观察脱氧胆酸(DCA)对AR42J胰腺腺泡细胞的损伤作用并探讨其对核转录因子(TF)活性的影响。方法 应用噻唑蓝(MTT)比色法检测DCA作用下细胞存活率改变,流式细胞术AV/PI双染法检测细胞的凋亡/坏死率。细胞经0.4mmoL/L DCA分别作用15 min、30 min、4h后收集培液上清,收集细胞并提取细胞质和细胞核蛋白,分别检测培液上清和胞质淀粉酶的活性,利用Luminex检测细胞核TF的DNA结合活性。结果 DCA对AR42J胰腺腺泡细胞的损伤作用呈浓度和时间依赖性,对细胞质内和培液中的淀粉酶水平无明显影响。在检测的40种TF活性变化中,DCA诱导ATF2、AR33、STAT5、NFAT、FKHR和NKX-2.5这6种TF活性明显升高,而RUNX/AML、NF-Y、MEF2和E2F1这4种TF活性则明显下降,其余30种TF活性无明显变化。结论 DCA对腺泡细胞的损伤作用主要表现为凋亡和坏死,对细胞内酶的合成和分泌功能没有明显影响。DCA诱导细胞核TF活性的变化,可能是其诱导细胞损伤的分子生物学基础。%Objective To study the cytotoxic effect of desoxycholic acid (DCA) on pancreatic acinar cells AR42J, its impact on the synthesis and secretion function of amylase, and the influence on the activity of nuclear transcription factor (TF). MethodsThe cytotoxic effect of DCS was detected in rat AR42J cells by using methyl thiazol tetrazolium (MTT) assay. The rate of apoptosis or necrosis was determined by flow cytometry. After the cells were incubated with DCA (0. 4 mmol/L) for 15 min, 30 min, or 4 h, the medium was collected to detect the activity of amylase. The cytoplamic protein was extracted to detect the activity of amylase, and nuclear protein was extracted to detect the DNA binding activity of 40 TFs by Luminex. Results DCA exerted cytotoxic effects on AR42J cells in a time-and dose-dependent manner, and induced cell apoptosis and necrosis. DCA had no significant influence on the amylase synthesis and secretion

  7. Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

    Directory of Open Access Journals (Sweden)

    Coreen Johnson

    Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

  8. Osmotic Effects on the Electrical Properties of Arabidopsis Root Hair Vacuoles in Situ1

    Science.gov (United States)

    Lew, Roger R.

    2004-01-01

    To assess the role of the vacuole in responses to hyperosmotic and hypo-osmotic stress, the electrical properties of the vacuole were measured in situ. A double-barrel micropipette was inserted into the vacuole for voltage clamping. A second double-barrel micropipette was inserted into the cytoplasm to provide a virtual ground that separated the electrical properties of the vacuole from those of the plasma membrane. Osmotic stress causes immediate electrical responses at the plasma membrane (Lew RR [1996] Plant Physiol 97: 2002-2005) and ion flux changes and turgor recovery (Shabala SN, Lew RR [2002] 129: 290-299) in Arabidopsis root cells. In situ, the vacuole also responds rapidly to changes in extracellular osmotic potential. Hyperosmotic treatment caused a very large increase in the ionic conductance of the vacuole. Hypo-osmotic treatment did not affect the vacuolar conductance. In either case, the vacuolar electrical potential was unchanged. Taken in concert with previous studies of changes at the plasma membrane, these results demonstrate a highly coordinated system in which the vacuole and plasma membrane are primed to respond immediately to hyperosmotic stress before changes in gene expression. PMID:14730070

  9. Phytochelatin-metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis.

    Science.gov (United States)

    Song, Won-Yong; Mendoza-Cózatl, David G; Lee, Youngsook; Schroeder, Julian I; Ahn, Sang-Nag; Lee, Hyun-Sook; Wicker, Thomas; Martinoia, Enrico

    2014-05-01

    Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

  10. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [3H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  11. Saccharomyces cerevisiae depend on vesicular traffic between Golgi and vacuole when Inositolphosphorylceramide synthase Aur1 is inactivated

    DEFF Research Database (Denmark)

    Voynova, Natalia S; Roubaty, Carole; Vazquez, Hector M;

    2015-01-01

    that vesicle mediated transport between Golgi, endosomes and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and Quinacrine uptake into vacuoles shows that Ab...

  12. The Expression of VacA in BCF of Helicobacter Pylori and Its Relationship to Vacuolated Effect

    Institute of Scientific and Technical Information of China (English)

    施理; 侯晓华; 易粹琼; 张锦坤

    2002-01-01

    Summary: The vacuolated effect of Helicobacter (H. Pylori) and its relationship to vacuolated cyto toxin antigen (VacA) were investigated by the method of cytotoxic test and SDS-pobyacrylamide gel electrophoresis (SDS-PAGE). Of the 62 clinical isolates, the broth culture filter (BCF) of 43 strains causecl the Vero cell intracytoplasmically vacuolated. H. Pylori strains were divided into H. Pylori (Toxin+) group with vacuolated effect and H. Pylori (Toxin-) group without vacuolated effect. The analysis of the BCF of H. Pylori (Toxin+) and that of H. Pylori (Toxin-) was studied by SDS-PAGE and Scan reader. A kind of protein with 87 ku molecular weight was recognized in the BCF of 30.23 % (13/43) H. Pylori (Toxin+) strains but in none of that of H. Pylori (Toxin-) strains, the difference was statistically significant (P<0. 05). There was a significant and concordant relation ship between OD of the protein band with 87 ku molecular weight and titer of vacuolated activity of H. Pylori(Toxin+) (r=0. 67 and P<0. 05 by linear regression analysis). H. Pylori strains were di-vided into H. Pylori (Toxin+) group with vacuolated effect and H. Pylori (Toxin-) group without vacuolated effect. The vacuolated effect of H. Pylori (Toxin+) was caused by the protein with 87 ku molecular weight (VacA).

  13. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

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    Kendi Okuda

    2016-06-01

    Full Text Available 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.

  14. THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

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    S. V. Isayenkov

    2013-06-01

    Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

  15. Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

    Science.gov (United States)

    Liu, Xin; Pitarresi, Jason R; Cuitiño, Maria C; Kladney, Raleigh D; Woelke, Sarah A; Sizemore, Gina M; Nayak, Sunayana G; Egriboz, Onur; Schweickert, Patrick G; Yu, Lianbo; Trela, Stefan; Schilling, Daniel J; Halloran, Shannon K; Li, Maokun; Dutta, Shourik; Fernandez, Soledad A; Rosol, Thomas J; Lesinski, Gregory B; Shakya, Reena; Ludwig, Thomas; Konieczny, Stephen F; Leone, Gustavo; Wu, Jinghai; Ostrowski, Michael C

    2016-09-01

    The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts. PMID:27633013

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

    Science.gov (United States)

    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

    2015-02-17

    Activation of an apical Ca(2+)-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(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-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, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.

  17. 糖尿病/星型胶质细胞富集磷蛋白酶-15的表达对急性胰腺炎腺泡细胞凋亡的影响%Effects of diabetes mellitus/astrocyte enriched phosphorus protease 15 expression on the apoptosis of acinar cells in acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    胡明星; 王玉柱; 刘传江; 秦涛

    2015-01-01

    目的 观察糖尿病/星型胶质细胞富集磷蛋白酶-15(PED/PEA-15)的表达对蛙皮素诱导的体外急性胰腺炎(AP)模型腺泡细胞凋亡的影响.方法 建立体外AP模型:用10 nmol/L的蛙皮素处理胰腺腺泡细胞株(AR42J细胞)建立AP模型,4h后检测培养液淀粉酶水平,实时荧光定量反转录聚合酶链反应(RT-PCR)检测PED/PEA-15的mRNA表达,吖啶橙/溴乙锭(AO/EB)法检测AR42J细胞的凋亡.构建PED/PEA-15的真核表达载体pcDNA3和空载体,利用脂质体转染AR42J细胞,72 h后加入10 nmol/L的蛙皮素处理,RT-PCR检测PED/PEA-15的mRNA表达水平,AO/EB法检测AR42J细胞的凋亡.结果 蛙皮素处理的AR42J细胞培养液淀粉酶水平较未处理细胞明显升高[(748.75±42.90) U/L较(249.75±27.16) U/L,P<0.05],PED/PEA-15的mRNA表达降低(5.24±0.66)倍(P<0.05),细胞凋亡率[(78.75±0.03)%]较未处理组[(17.50±0.04)%]升高(P<0.05).PED/PEA-15-pcDNA组AR42J细胞较空载体组PED/PEA-15的mRNA表达升高(4.27±0.78)倍(P<0.05),淀粉酶水平[(528.71±34.92) U/L]较空载体组[(856.29±52.39) U/L]明显下降(P<0.05),细胞凋亡率[(22.19±1.21)%]较空载体组[(68.92±1.83)%]降低(P<0.05),转染空载体组各项指标与未转染组差异无统计学意义(P>0.05).结论 PED/PEA-15蛋白在胰腺腺泡细胞的表达升高,能够抑制蛙皮素诱导的AR42J胰腺腺泡细胞凋亡.%Objective To investigate the effect of diabetes mellitus/astrocyte enriched phosphorus protease 15 (PED/PEA-15) expression on the apoptosis of acinar cells in bombesin-induced acute pancreatitis.Methods A model of acute pancreatitis with bombesin of 10 nmol/L was established.Amylase was determined 4 h later.The real-time reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of PED/PEA-15 mRNA.The pcDNA or empty vehicle was created and transfected into AR42J cells.After the AR42J cells were treated with 10 nmol/L bombesin for 72 h, the

  18. Cell therapy for salivary gland regeneration.

    Science.gov (United States)

    Lin, C-Y; Chang, F-H; Chen, C-Y; Huang, C-Y; Hu, F-C; Huang, W-K; Ju, S-S; Chen, M-H

    2011-03-01

    There are still no effective therapies for hyposalivation caused by irradiation. In our previous study, bone marrow stem cells can be transdifferentiated into acinar-like cells in vitro. Therefore, we hypothesized that transplantation with bone marrow stem cells or acinar-like cells may help functional regeneration of salivary glands. Bone marrow stem cells were labeled with nanoparticles and directly co-cultured with acinar cells to obtain labeled acinar-like cells. In total, 140 severely combined immune-deficiency mice were divided into 4 groups for cell therapy experiments: (1) normal mice, (2) mice receiving irradiation around their head-and-neck areas; (3) mice receiving irradiation and intra-gland transplantation with labeled stem cells; and (4) mice receiving irradiation and intra-gland transplantation with labeled acinar-like cells. Our results showed that salivary glands damaged due to irradiation can be rescued by cell therapy with either bone marrow stem cells or acinar-like cells for recovery of saliva production, body weight, and gland weight. Transdifferentiation of bone marrow stem cells into acinar-like cells in vivo was also noted. This study demonstrated that cell therapy with bone marrow stem cells or acinar-like cells can help functional regeneration of salivary glands, and that acinar-like cells showed better therapeutic potentials than those of bone marrow stem cells.

  19. Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

    Directory of Open Access Journals (Sweden)

    Jorge E Vidal

    2009-02-01

    Full Text Available OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: 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. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+ y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-. El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un

  20. ER and vacuoles: never been closer

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    Corrado eViotti

    2014-02-01

    Full Text Available The endoplasmic reticulum (ER represents the gateway for intracellular trafficking of membrane proteins, soluble cargoes and lipids. In all eukaryotes, the best described mechanism of exiting the ER is via COPII-coated vesicles, which transport both membrane proteins and soluble cargo to the cis-Golgi. The vacuole, together with the plasma membrane, is the most distal point of the secretory pathway, and many vacuolar proteins are transported from the ER through intermediate compartments. However, past results and recent findings demonstrate the presence of alternative transport routes from the ER towards the tonoplast, which are independent of Golgi- and post-Golgi trafficking. Moreover, the transport mechanism of the vacuolar proton pumps VHA-a3 and AVP1 challenges the current model of vacuole biogenesis, pointing to the endoplasmic reticulum for being the main membrane source for the biogenesis of the plant lytic compartment. This review gives an overview of the current knowledge on the transport routes towards the vacuole and discusses the possible mechanism of vacuole biogenesis in plants.

  1. Alteration of SH-group Contents in Red Beet Roots and Vacuoles under Osmotic Stress

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    Ozolina, N. V.

    2013-02-01

    Full Text Available The content of sulfhydryl groups in homogenate of red beets and isolated vacuoles under the conditions of osmotic stress was determined. It was demonstrated that the common content of sulfhydryl groups in the isolated vacuoles was 2.4 times higher, than in the homogenate. Under the conditions of osmotic stress, it was primarily denoted the reduction of common content of sulfhydryl groups in homogenate and in the isolated vacuoles. The most interesting results were obtained in determination of correlations between protein and non-protein SH-groups. Under the conditions of osmotic stress, while the contents of non-protein SH-groups in the isolated vacuoles was reduced, non-protein SH-groups in homogenate was greatly increased. This may be explained by the influx of the substances containing SH-groups out of vacuoles. Obtained results allow us to conclude that vacuoles play an important role in plant cell antioxidant processes and in maintenance of intracellular redox-homeostasis.

  2. PX domain and CD domain play different roles in localization and vacuolation of Sorting Nexin 10

    Institute of Scientific and Technical Information of China (English)

    YAO Dong; WU Bin; QIN BaoMing; PEI DuanQing

    2009-01-01

    Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting,trafficking and signal transduction.The PX domains of SNXs can bind to various phosphorelated phosphoinositides (Pls) and target the host proteins to endosomes.Recently,we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles.In this study,we aimed to identify regions in SNX10 critical for the vacuolation activity.We found that both the PX domain and the CD1 region were essential for vacuolation.We provided evidence that the PX domain was able to specifically bind to Ptdlns(3)P and target SNX10 to endosomes.A mutation in the β1 region of the PX domain (V15A) disrupted the Ptdlns(3)P binding ability and the endosomal localization of SNX10.However,correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles.We found that the CD1 region,which was not required for the localization,was indispensable for the vacuolation activity of SNX10.In summary,both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.

  3. A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

    Indian Academy of Sciences (India)

    Entsar Saheb; Ithay Biton; Katherine Maringer; John Bush

    2013-09-01

    Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.

  4. Primary Acinic Cell Carcinoma of the Breast: A Clinicopathological and Immunohistochemical Study

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    Kiyoshi Shingu

    2013-01-01

    Full Text Available Acinic cell carcinoma of the breast is an extremely rare, malignant neoplasm characterized by widespread acinar cell-like differentiation and clinically low-grade malignancy. Herein, we report a case of acinic cell carcinoma of the breast in a 41-year-old woman. The tumor was poorly demarcated but had a firm consistency. It was removed with lumpectomy, and sentinel lymph node biopsy was performed to check for metastasis. Microscopically, the tumor showed an infiltrative growth pattern with a combination of solid, trabecular, and microglandular areas. Many of the tumor cells had abundant clear vacuolated cytoplasm containing zymogen-typed granules which resemble acinar cells of the salivary glands. The immunohistochemical profile of the tumor was also similar to that of salivary gland acinic cell carcinoma: the tumor cells were positive for amylase, lysozyme, α-1-antichymotrypsin, S-100 protein, and epithelial membrane antigen and negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. She received postoperative chemoradiation therapy and has been well for 3 years since surgery. As studies on large series are lacking, further studies are needed to elucidate the biological characteristics of acinic cell carcinoma of the breast.

  5. Intratumoral injection of taxol in vivo suppresses A549 tumor showing cytoplasmic vacuolization.

    Science.gov (United States)

    Wang, Chaoyang; Chen, Tongsheng

    2012-04-01

    Based on our recent in vitro studies, this report was designed to explore the mechanism by which high concentration of taxol (70 µM) induced paraptosis-like cell death in human lung carcinoma (A549) cells, and to evaluate the therapeutic efficacy of taxol using A549 tumor-bearing mice in vivo. Exposure of cells to taxol induced time-dependent cytotoxicity and cytoplasmic vacuolization without the involvement of Bax, Bak, Mcl-1, Bcl-XL, and caspase-3. Although taxol treatment induced activating transcription factor 6 (ATF6) cleavage indicative of endoplasmic reticulum (ER) stress, silencing ATF6 by shATF6 did not prevent taxol-induced both cytotoxcity and cytoplasmic vacuolization, suggesting that taxol-induced cytoplasmic vacuolization and cell death were not due to ER stress. Moreover, taxol-treated cells did not show DNA fragmentation and loss of mitochondrial membrane potential, the typical characteristics of apoptosis. In addition, taxol-induced cytoplasmic vacuolization did not show the cellular lysis, the characteristics of oncosis, and positive of β-galactosidase, the characteristic of senescence, indicating that taxol induced paraptosis-like cell death is neither oncosis nor senescence. Moreover, our in vivo data showed that intratumoral injection of taxol (50 mg/kg) in A549 tumor xenograft mice on day 1 and day 19 potently suppressed tumor growth showing significant ER vacuolization without toxicity. In conclusion, high concentration of taxol exhibits a significant anticancer activity by inducing paraptosis-like cell death in vitro and in vivo, without significant toxicity, suggesting a promising therapeutic strategy for apoptosis-resistance cancer by inducing ER vacuolization.

  6. Redox Enzymes of Red Beetroot Vacuoles (Beta vulgaris L.

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    E.V. Pradedova

    2014-12-01

    Full Text Available Years of research have shown that some of the redox elements (enzymes, coenzymes, and co-substrate are isolated from each other kinetic and spatial manner (compartmentalization in the eukaryotic cells. The redox elements forming the "highly" and "widely" specialized redox system are found in all cell structures: mitochondria, plastids, peroxisomes, apoplast, nucleus etc. In recent years the active involvement of the central vacuole in the maintenance of the plant cell redox homeostasis is discussed, actually the information about the vacuolar redox system is very small. The high-priority redox processes and "redox-specialization" of the vacuolar compartment are not known. We have begun a study of red beet-root vacuole redox systems (Beta vulgaris L. and have identified redox enzymes such as: phenol peroxidase (EC 1.11.1.7, superoxide dismutase (EC 1.15.1.1 and glutathione reductase (EC 1.8.1.7. This paper presents some of the characteristics of these enzymes and considers the probable ways of their functioning in vacuolar redox chains.

  7. Aggressive human neuroblastomas show a massive increase in the numbers of autophagic vacuoles and damaged mitochondria.

    Science.gov (United States)

    Samardzija, Gordana; Stevovic, Tamara Kravic; Djuricic, Slavisa; Djokic, Dragomir; Djurisic, Marina; Ciric, Darko; Martinovic, Tamara; Bumbasirevic, Vladimir; Vujic, Dragana

    2016-01-01

    Autophagy is activated in cancer cells in response to multiple stresses and has been demonstrated to promote tumor cell survival and drug resistance in neuroblastoma (NB). This study was conducted to analyze the ultrastructural features of peripheral neuroblastic tumors (pNTs) and identify the relation of the types of NTs, the proliferation rate, and MYCN gene amplification with a number of autophagic vacuoles. Our results indicate that aggressive human NBs show a massive increase in the number of autophagic vacuoles associated with proliferation rate and that alteration of the mitochondria might be an important factor for the induction of autophagy in NTs. PMID:27669398

  8. Saccharomyces cerevisiae Vacuole in Zinc Storage and Intracellular Zinc Distribution▿ ‡

    OpenAIRE

    Simm, Claudia; Lahner, Brett; Salt, David; LeFurgey, Ann; Ingram, Peter; Yandell, Brian; Eide, David J.

    2007-01-01

    Previous studies of the yeast Saccharomyces cerevisiae indicated that the vacuole is a major site of zinc storage in the cell. However, these studies did not address the absolute level of zinc that was stored in the vacuole nor did they examine the abundances of stored zinc in other compartments of the cell. In this report, we describe an analysis of the cellular distribution of zinc by use of both an organellar fractionation method and an electron probe X-ray microanalysis. With these method...

  9. Glutathione Reductase of Vacuole. Comparison of Glutathione Reductase Activity of Vacuole and Tissue Extract of Red Beet Root (Beta vulgaris L.

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    E.V. Pradedova

    2016-02-01

    Full Text Available Glutathione reductase (GR, EC 1.8.1.7 is the enzyme that reduces oxidized glutathione (GSSG and thus regulates the redox state of glutathione (GSH/GSSG. GR has been studied in most plants. This enzyme has been identified in chloroplasts and cytosol, so these cellular compartments are considered to be the main place of the enzyme localization. In the same time, just a little is known about GR vacuoles. There are no conclusive evidences to prove the presence or absence of this enzyme in the vacuoles. GR activity was found in the vacuoles of red beet root cells (Beta vulgaris L.. The level of activity, the optimum pH and isoenzyme composition of GR were compared in the vacuoles and tissue extract of beet root. Vacuolar GR activity was quite high, it was 1.5-2 times higher than the activity of the tissue extract. Enzyme pH optimum of all the objects were identical. pH-optimum depend on the pyridine nucleotide nature: pH 7.0-8.0 was an optimal range with NADPH; pH 5.0 – with NADH. GR activity of the vacuoles and tissue extracts decreased in the presence of a noncompetitive inhibitor 1-chloro-2.4-dinitrobenzene (CDNB, indicating the specificity of this enzymatic reaction. Two bands with glutathione reductase activity have been identified in the vacuoles and tissue extracts using zymography method to determine the enzymatic activity in PAAG after electrophoresis of proteins. Belonging to the GR isoforms of these bands was confirmed by enzyme immunoassay (Western blotting. The electric mobility of isoforms of the study objects did not differ significantly. It is concluded that the biochemical characteristics of vacuolar glutathione reductase were substantially identical to the biochemical characteristics of other localization GR.

  10. Purification and proteomics of pathogen-modified vacuoles and membranes

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    Jo-Ana eHerweg

    2015-06-01

    Full Text Available 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.

  11. Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

    International Nuclear Information System (INIS)

    There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

  12. Natamycin Inhibits Vacuole Fusion at the Priming Phase via a Specific Interaction with Ergosterol▿

    Science.gov (United States)

    te Welscher, Yvonne Maria; Jones, Lynden; van Leeuwen, Martin Richard; Dijksterhuis, Jan; de Kruijff, Ben; Eitzen, Gary; Breukink, Eefjan

    2010-01-01

    The antifungal antibiotic natamycin belongs to the family of polyene antibiotics. Its antifungal activity arises via a specific interaction with ergosterol in the plasma membrane (te Welscher et al., J. Biol. Chem. 283:6393-6401, 2008). However, this activity does not involve disruption of the membrane barrier function, a well-known property of other members of the polyene antibiotic family, such as filipin and nystatin. Here we tested the effect of natamycin on vacuole membrane fusion, which is known to be ergosterol dependent. Natamycin blocked the fusion of isolated vacuoles without compromising the barrier function of the vacuolar membrane. Sublethal doses of natamycin perturbed the cellular vacuole morphology, causing the formation of many more small vacuolar structures in yeast cells. Using vacuoles isolated from yeast strains deficient in the ergosterol biosynthesis pathway, we showed that the inhibitory activity of natamycin was dependent on the presence of specific chemical features in the structure of ergosterol that allow the binding of natamycin. We found that natamycin inhibited the priming stage of vacuole fusion. Similar results were obtained with nystatin. These results suggest a novel mode of action of natamycin and perhaps all polyene antibiotics, which involves the impairment of membrane fusion via perturbation of ergosterol-dependent priming reactions that precede membrane fusion, and they may point to an effect of natamycin on ergosterol-dependent protein function in general. PMID:20385867

  13. Natamycin inhibits vacuole fusion at the priming phase via a specific interaction with ergosterol.

    Science.gov (United States)

    te Welscher, Yvonne Maria; Jones, Lynden; van Leeuwen, Martin Richard; Dijksterhuis, Jan; de Kruijff, Ben; Eitzen, Gary; Breukink, Eefjan

    2010-06-01

    The antifungal antibiotic natamycin belongs to the family of polyene antibiotics. Its antifungal activity arises via a specific interaction with ergosterol in the plasma membrane (te Welscher et al., J. Biol. Chem. 283:6393-6401, 2008). However, this activity does not involve disruption of the membrane barrier function, a well-known property of other members of the polyene antibiotic family, such as filipin and nystatin. Here we tested the effect of natamycin on vacuole membrane fusion, which is known to be ergosterol dependent. Natamycin blocked the fusion of isolated vacuoles without compromising the barrier function of the vacuolar membrane. Sublethal doses of natamycin perturbed the cellular vacuole morphology, causing the formation of many more small vacuolar structures in yeast cells. Using vacuoles isolated from yeast strains deficient in the ergosterol biosynthesis pathway, we showed that the inhibitory activity of natamycin was dependent on the presence of specific chemical features in the structure of ergosterol that allow the binding of natamycin. We found that natamycin inhibited the priming stage of vacuole fusion. Similar results were obtained with nystatin. These results suggest a novel mode of action of natamycin and perhaps all polyene antibiotics, which involves the impairment of membrane fusion via perturbation of ergosterol-dependent priming reactions that precede membrane fusion, and they may point to an effect of natamycin on ergosterol-dependent protein function in general. PMID:20385867

  14. Molecular Composition of Plant Vacuoles: Important but Less Understood Regulations and Roles of Tonoplast Lipids

    Directory of Open Access Journals (Sweden)

    Chunhua Zhang

    2015-06-01

    Full Text Available The vacuole is an essential organelle for plant growth and development. It is the location for the storage of nutrients; such as sugars and proteins; and other metabolic products. Understanding the mechanisms of vacuolar trafficking and molecule transport across the vacuolar membrane is of great importance in understanding basic plant development and cell biology and for crop quality improvement. Proteins play important roles in vacuolar trafficking; such proteins include Rab GTPase signaling proteins; cargo recognition receptors; and SNAREs (Soluble NSF Attachment Protein Receptors that are involved in membrane fusion. Some vacuole membrane proteins also serve as the transporters or channels for transport across the tonoplast. Less understood but critical are the roles of lipids in vacuolar trafficking. In this review, we will first summarize molecular composition of plant vacuoles and we will then discuss our latest understanding on the role of lipids in plant vacuolar trafficking and a surprising connection to ribosome function through the study of ribosomal mutants.

  15. Pathogen vacuole purification from legionella-infected amoeba and macrophages.

    Science.gov (United States)

    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.

  16. Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes

    NARCIS (Netherlands)

    Sachse, M.; Urbé, S.; Oorschot, V.; Strous, G.J.; Klumperman, J.

    2002-01-01

    In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron miscroscopy we show that the coat contains cla

  17. Thioploca spp: filamentous sulfur bacteria with nitrate vacuoles

    DEFF Research Database (Denmark)

    Jørgensen, BB; Gallardo, VA

    1999-01-01

    Thioploca spp. are multicellular, filamentous, colorless sulfur bacteria inhabiting freshwater and marine sediments. They have elemental sulfur inclusions similar to the phylogenetically closely related Beggiatoa, but in contrast to these they live in bundles surrounded by a common sheath. Vast...... communities of large Thioploca species live along the Pacific coast of South America and in other upwelling areas of high organic matter sedimentation with bottom waters poor in oxygen and rich in nitrate. Each cell of these thioplocas harbors a large liquid vacuole which is used as a storage for nitrate...... with a concentration of lip to 506 mM. The nitrate is used as an electron acceptor for sulfide oxidation and the bacteria may grow autotrophically or mixotrophically using acetate or other organic molecules as carbon source. The filaments stretch up into the overlying seawater, from which they take up nitrate...

  18. Bifurcation of the endocytic pathway into Rab5-dependent and -independent transport to the vacuole

    Science.gov (United States)

    Toshima, Junko Y.; Nishinoaki, Show; Sato, Yoshifumi; Yamamoto, Wataru; Furukawa, Daiki; Siekhaus, Daria Elisabeth; Sawaguchi, Akira; Toshima, Jiro

    2014-03-01

    The yeast Rab5 homologue, Vps21p, is known to be involved both in the vacuolar protein sorting (VPS) pathway from the trans-Golgi network to the vacuole, and in the endocytic pathway from the plasma membrane to the vacuole. However, the intracellular location at which these two pathways converge remains unclear. In addition, the endocytic pathway is not completely blocked in yeast cells lacking all Rab5 genes, suggesting the existence of an unidentified route that bypasses the Rab5-dependent endocytic pathway. Here we show that convergence of the endocytic and VPS pathways occurs upstream of the requirement for Vps21p in these pathways. We also identify a previously unidentified endocytic pathway mediated by the AP-3 complex. Importantly, the AP-3-mediated pathway appears mostly intact in Rab5-disrupted cells, and thus works as an alternative route to the vacuole/lysosome. We propose that the endocytic traffic branches into two routes to reach the vacuole: a Rab5-dependent VPS pathway and a Rab5-independent AP-3-mediated pathway.

  19. Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Feddersen, Søren; Christiansen, Janne K;

    2004-01-01

    In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide......-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and...

  20. Differential Induction of Cytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones.

    Science.gov (United States)

    Trabbic, Christopher J; Dietsch, Heather M; Alexander, Evan M; Nagy, Peter I; Robinson, Michael W; Overmeyer, Jean H; Maltese, William A; Erhardt, Paul W

    2014-01-01

    Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of non-apoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity, but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis. PMID:24527179

  1. Grape berry vacuole : a complex and heterogeneous membrane system specialized in the accumulation of solutes

    OpenAIRE

    Fontes, N.; Gerós, H.; Delrot, Serge

    2011-01-01

    Vacuoles fulfill highly specialized functions depending on cell type and tissue and plant developmental stage. This complex and dynamic organelle is the main reservoir of grape berry cells, playing a major role during fruit development and ripening. Berry development is accompanied by modifications in size, composition, color, texture, flavor, and pathogen susceptibility, primarily because of changes in vacuolar content. Most aroma and flavor compounds are not evenly distributed in the berry,...

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

    OpenAIRE

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

    2008-01-01

    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 monola...

  3. Alkalinity of neutrophil phagocytic vacuoles is modulated by HVCN1 and has consequences for myeloperoxidase activity.

    Science.gov (United States)

    Levine, Adam P; Duchen, Michael R; de Villiers, Simon; Rich, Peter R; Segal, Anthony W

    2015-01-01

    The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH. PMID:25885273

  4. Loss of the BRCA1-interacting helicase BRIP1 results in abnormal mammary acinar morphogenesis.

    Directory of Open Access Journals (Sweden)

    Kazuhiro Daino

    Full Text Available BRIP1 is a DNA helicase that directly interacts with the C-terminal BRCT repeat of the breast cancer susceptibility protein BRCA1 and plays an important role in BRCA1-dependent DNA repair and DNA damage-induced checkpoint control. Recent studies implicate BRIP1 as a moderate/low-penetrance breast cancer susceptibility gene. However, the phenotypic effects of BRIP1 dysfunction and its role in breast cancer tumorigenesis remain unclear. To explore the function of BRIP1 in acinar morphogenesis of mammary epithelial cells, we generated BRIP1-knockdown MCF-10A cells by short hairpin RNA (shRNA-mediated RNA interference and examined its effect in a three-dimensional culture model. Genome-wide gene expression profiling by microarray and quantitative RT-PCR were performed to identify alterations in gene expression in BRIP1-knockdown cells compared with control cells. The microarray data were further investigated using the pathway analysis and Gene Set Enrichment Analysis (GSEA for pathway identification. BRIP1 knockdown in non-malignant MCF-10A mammary epithelial cells by RNA interference induced neoplastic-like changes such as abnormal cell adhesion, increased cell proliferation, large and irregular-shaped acini, invasive growth, and defective lumen formation. Differentially expressed genes, including MCAM, COL8A1, WIPF1, RICH2, PCSK5, GAS1, SATB1, and ELF3, in BRIP1-knockdown cells compared with control cells were categorized into several functional groups, such as cell adhesion, polarity, growth, signal transduction, and developmental process. Signaling-pathway analyses showed dysregulation of multiple cellular signaling pathways, involving LPA receptor, Myc, Wnt, PI3K, PTEN as well as DNA damage response, in BRIP1-knockdown cells. Loss of BRIP1 thus disrupts normal mammary morphogenesis and causes neoplastic-like changes, possibly via dysregulating multiple cellular signaling pathways functioning in the normal development of mammary glands.

  5. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

  6. 荧光蛋白在植物活细胞液泡成像中的应用研究进展%Recent Progress in Living Cell Imaging of Plant Vacuole Using Fluorescent-protein Transgenic Lines and Three-dimensional Imaging

    Institute of Scientific and Technical Information of China (English)

    叶庆亮; 曹建华

    2011-01-01

    高等植物细胞中,液泡在各种细胞信号转导事件和形态建成中起着重要的作用.这一细胞内部结构在分裂和分化期间其功能和形状不断地变化着.为分析这些连续的变化,绿色荧光蛋白(green fluorescent protein,GFP)及其他荧光蛋白活体标记技术普遍用来跟踪特异蛋白的定位及变动.为使液泡可视成像,有几种途径可用来选择适当的荧光蛋白融合伴侣,如液泡膜内在蛋白和构造蛋白相关蛋白等就非常适合应用于液泡成像.此外,三维重建法在定位细胞内广为分布的细胞器时也不可或缺.同时,等值面表面模化过程对液泡膜成像也非常有用.本文综述液泡的结构、种类和功能,并概括各种融合的绿色荧光蛋白在植物活细胞液泡成像中的应用及其三维成像技术的研究进展.%In high plant cells, vacuoles play important roles in a variety of cellular events, including cell division,morphogenesis, and signal transduction.These intracellular structures undergo dynamic changes in their shapes and functions during cell division and differentiation, and to analyze these sequential structural changes, the vital labeling technique, using the green-fluorescent protein or other fluorescent proteins, has commonly been used to follow the localization and translocation of specific proteins.To visualize vacuoles, the tonoplast-intrinsic proteins and syntaxin-related proteins are available for selecting the appropriate fluorescent-protein fusion partner for vacuolar imaging.In addition, three-dimensional reconstruction methods are indispensable for localizing the widely distributed organelles within the cell.The maximum intensity projection method is suitable for cytoskeletal structures, while contour-based surface modeling possesses many advantages for vacuolar membranes.In this article, we summarize the plant vacuoles and the recent progress in living cell imaging of the plant vacuoles using various fusions

  7. Unsteady diffusional screening in 3D pulmonary acinar structures: from infancy to adulthood.

    Science.gov (United States)

    Hofemeier, Philipp; Shachar-Berman, Lihi; Tenenbaum-Katan, Janna; Filoche, Marcel; Sznitman, Josué

    2016-07-26

    Diffusional screening in the lungs is a physical phenomenon where the specific topological arrangement of alveolated airways of the respiratory region leads to a depletion, or 'screening', of oxygen molecules with increasing acinar generation. Here, we revisit diffusional screening phenomena in anatomically-inspired pulmonary acinar models under realistic breathing maneuvers. By modelling 3D bifurcating alveolated airways capturing both convection and diffusion, unsteady oxygen transport is investigated under cyclic breathing motion. To evaluate screening characteristics in the developing lungs during growth, four representative stages of lung development were chosen (i.e. 3 months, 1 year and 9 months, 3 years and adulthood) that capture distinct morphological acinar changes spanning alveolarization phases to isotropic alveolar growth. Numerical simulations unveil the dramatic changes in O2 transport occurring during lung development, where young infants exhibit highest acinar efficiencies that rapidly converge with age to predictions at adulthood. With increased ventilatory effort, transient dynamics of oxygen transport is fundamentally altered compared to tidal breathing and emphasizes the augmented role of convection. Resolving the complex convective acinar flow patterns in 3D acinar trees allows for the first time a spatially-localized and time-resolved characterization of oxygen transport in the pulmonary acinus, from infancy to adulthood.

  8. DOG1: a novel marker of salivary acinar and intercalated duct differentiation.

    Science.gov (United States)

    Chênevert, Jacinthe; Duvvuri, Umamaheswar; Chiosea, Simion; Dacic, Sanja; Cieply, Kathleen; Kim, Jean; Shiwarski, Daniel; Seethala, Raja R

    2012-07-01

    Anoctamin-1 (ANO1) (DOG1, TMEM16a) is a calcium-activated chloride channel initially described in gastrointestinal stromal tumors, but now known to be expressed in a variety of normal and tumor tissues including salivary tissue in murine models. We herein perform a comprehensive survey of DOG1 expression in 156 cases containing non-neoplastic human salivary tissues and tumors. ANO1 mRNA levels were significantly higher (8-fold increase, Pcells, striated and excretory ducts were invariably negative. All acinic cell carcinomas (n=28) were DOG1 positive demonstrating a complex mixture of intense (3+) apical membranous, cytoplasmic and complete membranous staining. Most ductal tumor types were negative or only showed a subset of positive cases. Within the biphasic tumor category, adenoid cystic carcinomas (18/24 cases) and epithelial-myoepithelial carcinomas (8/15 cases) were frequently positive, often showing a distinctive combined apical ductal and membranous/cytoplasmic myoepithelial staining profile. Thus, DOG1 staining is a marker of salivary acinar and to a lesser extent intercalated duct differentiation. Strong staining can be used to support the diagnosis of acinic cell carcinoma. DOG1 may also be a marker of a 'transformed' myoepithelial phenotype in a subset of biphasic salivary gland malignancies.

  9. Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

    Directory of Open Access Journals (Sweden)

    Masahiro Nakamori

    Full Text Available BACKGROUND: Rimmed vacuoles (RVs are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM and distal myopathy with RVs (DMRV. Granulovacuolar degeneration (GVD bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers. METHODS: Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1 tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK], (2 lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1, and (3 other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43] in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization. RESULTS: GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs. CONCLUSIONS: These results suggest that RVs of muscle

  10. Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds.

    Science.gov (United States)

    Maeshima, M.; Hara-Nishimura, I.; Takeuchi, Y.; Nishimura, M.

    1994-01-01

    Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings. PMID:12232303

  11. Encapsulation of Living Leishmania Promastigotes in Artificial Lipid Vacuoles.

    Directory of Open Access Journals (Sweden)

    Carlos E S Guedes

    Full Text Available After phagocytosis by mammalian macrophages, promastigote forms of Leishmania parasites settle inside intracellular parasitophorous vacuoles (PVs in which they transform into amastigote forms and replicate. Here, using a variant of the 'inverted emulsion' method, we succeeded in encapsulating living L. amazonensis parasites in giant artificial liposomes that serve as model PVs. We were able to control the size of liposomes, the pH and the composition of their internal volume, and the number of internalized parasites per liposome. L. amazonensis promastigotes encapsulated in liposomes filled with RPMI-Dextran solution at pH 7.5 or 6.5 survived up to 96 h at 24°C. At 37°C and pH 5.5, parasites survived 48h. This method paves the way to identifying certain effectors secreted by the parasite and to unraveling specific mechanisms of fusion between the PV and intracellular vesicles of the host cell. This method will also facilitate the study of the temporal evolution of biophysical properties of the PV during its maturation.

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

    Science.gov (United States)

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

    2016-01-01

    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. PMID:27257019

  13. Recovery of pancreas from mild puromycin-induced injury. A histologic and ultrastructure study in rats.

    Science.gov (United States)

    Longnecker, D S; Crawford, B G; Nadler, D J

    1975-01-01

    Pancreatic acinar cells undergo degeneration or necrosis following injection of puromycin intraperitoneally in rats. The purpose of this study was to characterize recovery following injection of four hourly doses of puromycin, 40mg/kg of body weight, examining the pancreas histologically and by electron microscopy. The number of dividing acinar cells increased following injury. By 12 to 24 hours following treatment, electron microscopy showed numerous autophagic vacuoles and intracisternal granules in the cells. By 48 hours, these were largely cleared from surviving cells although the intracisternal granules persisted in isolated acinar cells as long as 144 hours. At 24 hours, there was debris in the acinar lumens and interstitial space. We conclude that some acinar cells injured by puromycin may survive and be restored to normal structure; that surviving acinar cells can extrude autophagic vacuoles; and that necrotic acinar cells are replaced by regeneration following puromycin-induced injury in rats. PMID:1111495

  14. Vacuole-targeting fungicidal activity of amphotericin B

    Directory of Open Access Journals (Sweden)

    Akira eOgita

    2012-03-01

    Full Text Available Invasive fungal infections are recognized as major threats to patients with immune depression as well as those with cancer chemotherapy. Amphotericin B (AmB, a classical antifungal agent with a polyene macrolide structure, is widely used for the control of serious fungal infections. However, the clinical use of this antibiotic is limited by the treatment-associated side effects and the appearance of resistant strains. AmB lethality has been generally elucidated by the alteration of plasma membrane ion permeability due to its specific binding to plasma membrane ergosterol. While, the recent studies with Saccharomyces cerevisiae and Candida albicans reveals the vacuole disruptive action as another cause of AmB lethality on the basis of its marked amplification in combination with allicin, an allyl sulfur compound from garlic. Indeed, AmB causes a serious structural damage to the vacuole membrane at a lethal concentration, and even at a non-lethal concentration in combination with allicin. Such an enhancement effect of allicin is dependent on an inhibition of ergosterol-trafficking from the plasma membrane to the vacuole membrane, which is considered to be a cellular response to protect against the vacuole membrane disintegration. Allicin can also decrease the minimum fungicidal concentration of AmB against the pathogenic fungi C. albicans and Aspergillus fumigatus, as is the case of S. cerevisiae. The synergistic fungicidal activities of AmB and allicin may have significant implications in the development of the vacuole-targeting chemotherapy against fungal infections.

  15. Vacuole import and degradation pathway:Insights into a specialized autophagy pathway

    Institute of Scientific and Technical Information of China (English)

    Abbas; A; Alibhoy; Hui-Ling; Chiang

    2011-01-01

    Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.

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

    Directory of Open Access Journals (Sweden)

    Samuel Leslie Palframan

    2012-07-01

    Full Text Available More than fifty percent of the world’s population is infected with Helicobacter pylori. Chronic infection with the Gram-negative pathogen is associated with the development of peptic ulcers and is linked to an increased risk of gastric cancer. H. pylori secrete 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 bipartite toxin, Vacuolating cytotoxin A (VacA. After secretion from the bacteria via a type V autotransport secretion system, the two VacA subunits (p33 and p55 enter host cells and cause severe vacuolation. This is 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 more 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 questions that need to be

  17. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

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    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

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

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    Kazunari Momma

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. Cell-Specific Cre Strains For Genetic Manipulation in Salivary Glands.

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    Eri O Maruyama

    Full Text Available The secretory acinar cells of the salivary gland are essential for saliva secretion, but are also the cell type preferentially lost following radiation treatment for head and neck cancer. The source of replacement acinar cells is currently a matter of debate. There is evidence for the presence of adult stem cells located within specific ductal regions of the salivary glands, but our laboratory recently demonstrated that differentiated acinar cells are maintained without significant stem cell contribution. To enable further investigation of salivary gland cell lineages and their origins, we generated three cell-specific Cre driver mouse strains. For genetic manipulation in acinar cells, an inducible Cre recombinase (Cre-ER was targeted to the prolactin-induced protein (Pip gene locus. Targeting of the Dcpp1 gene, encoding demilune cell and parotid protein, labels intercalated duct cells, a putative site of salivary gland stem cells, and serous demilune cells of the sublingual gland. Duct cell-specific Cre expression was attempted by targeting the inducible Cre to the Tcfcp2l1 gene locus. Using the R26Tomato Red reporter mouse, we demonstrate that these strains direct inducible, cell-specific expression. Genetic tracing of acinar cells using PipGCE supports the recent finding that differentiated acinar cells clonally expand. Moreover, tracing of intercalated duct cells expressing DcppGCE confirms evidence of duct cell proliferation, but further analysis is required to establish that renewal of secretory acinar cells is dependent on stem cells within these ducts.

  1. Cell-Specific Cre Strains For Genetic Manipulation in Salivary Glands.

    Science.gov (United States)

    Maruyama, Eri O; Aure, Marit H; Xie, Xiaoling; Myal, Yvonne; Gan, Lin; Ovitt, Catherine E

    2016-01-01

    The secretory acinar cells of the salivary gland are essential for saliva secretion, but are also the cell type preferentially lost following radiation treatment for head and neck cancer. The source of replacement acinar cells is currently a matter of debate. There is evidence for the presence of adult stem cells located within specific ductal regions of the salivary glands, but our laboratory recently demonstrated that differentiated acinar cells are maintained without significant stem cell contribution. To enable further investigation of salivary gland cell lineages and their origins, we generated three cell-specific Cre driver mouse strains. For genetic manipulation in acinar cells, an inducible Cre recombinase (Cre-ER) was targeted to the prolactin-induced protein (Pip) gene locus. Targeting of the Dcpp1 gene, encoding demilune cell and parotid protein, labels intercalated duct cells, a putative site of salivary gland stem cells, and serous demilune cells of the sublingual gland. Duct cell-specific Cre expression was attempted by targeting the inducible Cre to the Tcfcp2l1 gene locus. Using the R26Tomato Red reporter mouse, we demonstrate that these strains direct inducible, cell-specific expression. Genetic tracing of acinar cells using PipGCE supports the recent finding that differentiated acinar cells clonally expand. Moreover, tracing of intercalated duct cells expressing DcppGCE confirms evidence of duct cell proliferation, but further analysis is required to establish that renewal of secretory acinar cells is dependent on stem cells within these ducts.

  2. The pathogen-occupied vacuoles of Anaplasma phagocytophilum and Anaplasma marginale interact with the endoplasmic reticulum

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    Hilary Kay Truchan

    2016-03-01

    Full Text Available 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

  3. Hyperacidification of Vacuoles by the Combined Action of Two Different P-ATPases in the Tonoplast Determines Flower Color

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    Marianna Faraco

    2014-01-01

    Full Text Available The acidification of endomembrane compartments is essential for enzyme activities, sorting, trafficking, and trans-membrane transport of various compounds. Vacuoles are mildly acidic in most plant cells because of the action of V-ATPase and/or pyrophosphatase proton pumps but are hyperacidified in specific cells by mechanisms that remained unclear. Here, we show that the blue petal color of petunia ph mutants is due to a failure to hyperacidify vacuoles. We report that PH1 encodes a P3B-ATPase, hitherto known as Mg2+ transporters in bacteria only, that resides in the vacuolar membrane (tonoplast. In vivo nuclear magnetic resonance and genetic data show that PH1 is required and, together with the tonoplast H+ P3A-ATPase PH5, sufficient to hyperacidify vacuoles. PH1 has no H+ transport activity on its own but can physically interact with PH5 and boost PH5 H+ transport activity. Hence, the hyperacidification of vacuoles in petals, and possibly other tissues, relies on a heteromeric P-ATPase pump.

  4. The pattern of fibrosis in the acinar zone 3 areas in early alcoholic liver disease

    DEFF Research Database (Denmark)

    Junge, Jette; Horn, T; Vyberg, M;

    1991-01-01

    The degree of fibrosis and the pattern of collagen distribution in the acinar zone 3, as well as the thickness of the terminal hepatic vein walls (THV) were analyzed in 48 consecutive liver needle biopsies from 48 alcoholics with preserved liver architecture. The fibrosis occurred to more or less...

  5. Ectrodactyly and Lethal Pulmonary Acinar Dysplasia Associated with Homozygous FGFR2 Mutations Identified by Exome Sequencing.

    Science.gov (United States)

    Barnett, Christopher P; Nataren, Nathalie J; Klingler-Hoffmann, Manuela; Schwarz, Quenten; Chong, Chan-Eng; Lee, Young K; Bruno, Damien L; Lipsett, Jill; McPhee, Andrew J; Schreiber, Andreas W; Feng, Jinghua; Hahn, Christopher N; Scott, Hamish S

    2016-09-01

    Ectrodactyly/split hand-foot malformation is genetically heterogeneous with more than 100 syndromic associations. Acinar dysplasia is a rare congenital lung lesion of unknown etiology, which is frequently lethal postnatally. To date, there have been no reports of combinations of these two phenotypes. Here, we present an infant from a consanguineous union with both ectrodactyly and autopsy confirmed acinar dysplasia. SNP array and whole-exome sequencing analyses of the affected infant identified a novel homozygous Fibroblast Growth Factor Receptor 2 (FGFR2) missense mutation (p.R255Q) in the IgIII domain (D3). Expression studies of Fgfr2 in development show localization to the affected limbs and organs. Molecular modeling and genetic and functional assays support that this mutation is at least a partial loss-of-function mutation, and contributes to ectrodactyly and acinar dysplasia only in homozygosity, unlike previously reported heterozygous activating FGFR2 mutations that cause Crouzon, Apert, and Pfeiffer syndromes. This is the first report of mutations in a human disease with ectrodactyly with pulmonary acinar dysplasia and, as such, homozygous loss-of-function FGFR2 mutations represent a unique syndrome. PMID:27323706

  6. Geometrical influence of pulmonary acinar models on respiratory flows and particle deposition

    Science.gov (United States)

    Hofemeier, Philipp; Sznitman, Josue

    2012-11-01

    Due to experimental challenges in assessing respiratory flows in the deep regions of the lungs, computational simulations are typically sought to quantify inhaled aerosol transport and deposition in the acinus. Most commonly, simulations are performed using generic geometries of alveoli, including spheres, toroids and polyhedra to mimic the acinar region. However, local respiratory flows and ensuing particle trajectories are anticipated to be highly influenced by the specific geometrical structures chosen. To date, geometrical influences have not yet been thoroughly quantified. Knowing beforehand how geometries affect acinar flows and particle transport is critical in translating simulated data to predictions of aerosol deposition in real lungs. Here, we conduct a systematic investigation on a number of generic acinar models. Simulations are conducted for simple alveolated airways featuring a selection of geometries. Deposition patterns and efficiencies are quantified both for massless particles, highlighting details of the local flow, and micron-scale aerosols. This latter group of particles represents an important class of inhaled aerosols known to reach and deposit in the acinus. Our work emphasizes the subtleties of acinar geometry in determining the fate of inhaled aerosols.

  7. Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

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    Anton R Dluzewski

    Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

  8. Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion

    NARCIS (Netherlands)

    M.K. Singh; F. Krüger; H. Beckmann; S. Brumm; J.E.M. Vermeer; T. Munnik; U. Mayer; Y.D. Stierhof; C. Grefen; K. Schumacher; G. Jürgens

    2014-01-01

    Plasma-membrane proteins such as ligand-binding receptor kinases, ion channels, or nutrient transporters are turned over by targeting to a lytic compartment--lysosome or vacuole--for degradation. After their internalization, these proteins arrive at an early endosome, which then matures into a late

  9. The Rice RMR1 Associates with a Distinct Prevacuolar Compartment for the Protein Storage Vacuole Pathway

    Institute of Scientific and Technical Information of China (English)

    Yun Shen; Junqi Wang; Yu Ding; SzeWan Lo; Guillaume Gouzerh; Jean-Marc Neuhaus; Liwen Jiang

    2011-01-01

    Transport of vacuolar proteins from Golgi apparatus or trans-Golgi network (TGN) to vacuoles is a receptormediated process via an intermediate membrane-bound prevacuolar compartment (PVC) in plant cells.Both vacuolar sorting receptor (VSR) and receptor homology region-transmembrane domain-RING-H2 (RMR) proteins have been shown to function in transporting storage proteins to protein storage vacuole (PSV),but little is known about the nature of the PVC for the PSV pathway.Here,we use the rice RMR1 (OsRMR1) as a probe to study the PSV pathway in plants.Immunogold electron microscopy (EM) with specific OsRMR1 antibodies showed that OsRMR1 proteins were found in the Golgi apparatus,TGN,and a distinct organelle with characteristics of PVC in both rice culture cells and developing rice seeds,as well as the protein body type Ⅱ (PBII) or PSV in developing rice seeds.This organelle,also found in both tobacco BY-2 and Arabidopsis suspension cultured cells,is morphologically distinct from the VSR-positive multivesicular lytic PVC or multivesicular body (MVB) and thus represent a PVC for the PSV pathway that we name storage PVC (sPVC).Further in vivo and in vitro interaction studies using truncated OsRMR1 proteins secreted into the culture media of transgenic BY-2 suspension cells demonstrated that OsRMR1 functions as a sorting receptor in transporting vicilin-like storage proteins.

  10. Parasitophorous vacuole membrane of Plasmodium knowlesi

    Energy Technology Data Exchange (ETDEWEB)

    Nillni, E.A.; Wallach, D.F.H.

    1986-05-01

    The authors have evaluated the occurrence of host cell membrane protein and parasite protein in the vacuolar membrane (VM) of isolated parasites. Parasites were labeled by incorporation of (/sup 35/S)methionine and by lactoperoxidase-catalyzed /sup 125/I iodination. Of the two prominent /sup 125/I-labeled components, one, not detected by metabolic labeling corresponded in M/sub r/ to erythrocyte band 3 (90 kDa). Trypsinization of radioiodinated parasites for 5' or 20' yield a 35 kDa fragment, not seen in untreated samples and compatible with the trypsin degradation of band 3 from the cytoplasmic side. Tryptic peptide maps of the 35 kDa revealed a very acidic peptide corresponding to the highly anionic tryptic peptide of band 3 showed by others. The second prominent /sup 125/I-labeled VM protein had an M/sub r/ 74,000 corresponding to a protein metabolically labeled with (/sup 35/S)methionine, suggesting it is inserted into the VM by the parasites. Several less prominent proteins labeling with both (/sup 35/S)methionine and /sup 125/I were also detected (140 kDa, 55 kDa, 45 kDa). A faint /sup 125/I-labeled triple (220-230 kDa) is compatible with a trace amounts of spectrin, usually a prominent component of red cell membrane. The results indicate that host cell band 3 is a prominent component of the VM, but that this membrane also contains several parasite-synthesized proteins.

  11. Inhibition of eIF2α dephosphorylation inhibits ErbB2-induced deregulation of mammary acinar morphogenesis

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    Avivar-Valderas Alvaro

    2009-09-01

    Full Text Available Abstract Background The ErbB2/Her2/Neu receptor tyrosine kinase is amplified in ~30% of human breast cancers. Phosphorylation of the translation initiation factor, eIF2α inhibits global protein synthesis and activates a stress signaling and growth suppressive program. We have shown that forced phosphorylation of eIF2α can suppress head and neck, colorectal carcinoma and multiple myeloma tumor growth and/or survival. Here we explore whether ErbB2 modulates eIF2α phosphorylation and whether forced phosphorylation of the latter can antagonize ErbB2 deregulation of mammary acinar morphogenesis. Results We tested whether ErbB2 signaling influenced eIF2α signaling and whether enhanced phosphorylation of the latter affected ErbB2-deregulated mammary acinar development. We obtained stable MCF10A cells overexpressing wild-type (Wt Neu/ErbB2 or a constitutively active (CA variant via retroviral delivery or mammary tumor cells from MMTV-Neu tumors. Western blotting, RT-PCR and confocal microscopy were used to analyze the effects of ErbB2 activation on eIF2α signaling and the effect of the GADD34-PP1C inhibitor salubrinal. Wt- and MMTV-Neu cells formed aberrant acini structures resembling DCIS, while CA-ErbB2 overexpression induced invasive lesions. In these structures we found that CA-ErbB2 but not the Wt variant significantly down-regulated the pro-apoptotic gene CHOP. This occurred without apparent modulation of basal phosphorylation of PERK and eIF2α or induction of its downstream target ATF4. However, inhibition of eIF2α dephosphorylation with salubrinal was sufficient to inhibit Wt- and CA-ErbB2- as well as MMTV-Neu-induced deregulation of acinar growth. This was linked to enhanced CHOP expression, inhibition of proliferation, induction of apoptosis and luminal clearing in Wt-ErbB2 and to inhibition of cyclin D1 levels and subsequent proliferation in CA-ErbB2 cells. Conclusion Depending on the strength of ErbB2 signaling there is a differential

  12. Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability

    Institute of Scientific and Technical Information of China (English)

    Juan Sun; Kazuo Aoki; Jin-Xu Zheng; Bing-Zhong Su; Xiao-Hui Ouyang; Junichi Misumi

    2006-01-01

    AIM: To determine whether Helicobacter pylori (H pylori) vacuolating cytotoxin (VacA) regulates release of proinflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6)or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes.METHODS: Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial (AGS) cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays (ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR)were performed to examine the effects of Hpylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacA-induced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response.RESULTS: Addition of VacA alone reduced AGS cell viability (P< 0.05), and this reduction was enhanced by high doses of NaCl (P< 0.05). VacA alone induced expression of TNF-α, IL-8 and IL-1β, while NaCl alone induced expression of TNF-α and IL-1β. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-a, expression was dosedependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by ELISA. Positive immunohistochemical staining of IL- 1, IL-6, and TNF-αwas found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However,the staining of these three cytokines was stronger in H pylori-infected animals fed with a 5g/kg NaCl diet.CONCLUSION: VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines induced by Vac A, suggesting that NaCl plays an important role in Hpylori-induced gastric epithelial cell cytotoxicity.

  13. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

    Energy Technology Data Exchange (ETDEWEB)

    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 by 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.

  14. Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires

    Science.gov (United States)

    Wood, Rebecca E.; Newton, Patrice; Latomanski, Eleanor A.

    2015-01-01

    Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology. PMID:26216429

  15. Acinus-on-a-chip: a microfluidic platform for pulmonary acinar flows

    Science.gov (United States)

    Fishler, Rami; Mulligan, Molly; Sznitman, Josue; Sznitman Biofluids Team

    2013-11-01

    Convective respiratory flows in the pulmonary acinus and their influence on the fate of inhaled particles are typically studied using computational fluid dynamics (CFD) or scaled-up experimental models. However, current experiments generally capture only flow dynamics, without inhaled particle dynamics, due to difficulties in simultaneously matching flow and particle dynamics. In an effort to overcome these limitations, we have designed a novel microfluidic device mimicking acinar flow conditions directly at the physiological scale. The model features an anatomically-inspired acinar geometry with five dichotomously branching airway generations lined with periodically expanding and contracting alveoli. Using micro-particle image velocimetry (PIV), we reveal experimentally a gradual transition of alveolar flow patterns along the acinar tree from recirculating to radial streamlines, in support of previous predictions from CFD simulations. We demonstrate the applicability of the device for studying the mechanisms of particle deposition in the pulmonary acinus by mapping deposition sites of airborne fluorescent micro-particles (0.1-1 μm) and visualizing trajectories of airborne incense particles inside the system.

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

    International Nuclear Information System (INIS)

    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 125I-[Tyr1]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 Mg2+. When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125I-[Tyr1]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

  17. A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles.

    Directory of Open Access Journals (Sweden)

    Shira Ninio

    2009-01-01

    Full Text Available Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

  18. The influence of osmotic stress on the content of calcium ions in the red beet vacuoles and on the transport activity of tonoplast proton pumps

    Directory of Open Access Journals (Sweden)

    Ozolina N.V.

    2012-05-01

    Full Text Available The contents of calcium ions in the isolated vacuoles and in intact red beets under the conditions of dormancy and osmotic stress was determined. It is demonstrated that the content of calcium ions in the red beet vacuoles not exposed to osmotic stress makes 13.3% of the total content these ions in intact red beets. Under the conditions of osmotic stress, this indicator increases substantially. Furthermore, under the conditions of hyperosmotic stress, the content of calcium ions in the vacuoles was 30%, while under hypoosmotic stress it was 49% of the total content of these ions in the intact red beet. The transition of calcium ions from the cytoplasm and other compartments into the vacuole under the conditions of osmotic stress is, probably, one of forms of participation of the vacuole in adaptation processes of the plant cell under this kind of abiotic stress. It has been demonstrated for the first time that tonoplast proton pumps, which actively participate in provision of calcium homeostasis in cytoplasm, substantially activate their transport activity under osmotic stress, what allows one to speak about their important role in the cell’s protective programs. Under normal (no stress conditions, artificial elevation of the content of calcium ions led to inhibition of activity of the tonoplast proton pumps, while under gipoosmotic stress the activity of tonoplast proton pumps increased, what might aid to restoring homeoctasis with respect to calcium ions in cytoplasm.

  19. Coxiella burnetii and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses

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    Jess A Millar

    2015-08-01

    Full Text Available Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up- and 311 genes down-regulated in C. burnetii infected cells, whereas 698 genes (534 genes up- and 164 genes down-regulated were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (~7% are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs that were expressed in THP-1 cells and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections. Intriguingly, our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches.

  20. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection

    Science.gov (United States)

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires’ disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells. PMID:25496424

  1. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection.

    Science.gov (United States)

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires' disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells.

  2. Vacuolate-attached filaments: highly productive Ridgeia piscesae epibionts at the Juan de Fuca hydrothermal vents

    OpenAIRE

    Kalanetra, Karen M.; Nelson, Douglas C.

    2010-01-01

    Vacuolate sulfur bacteria with high morphological similarity to vacuolate-attached filaments previously described from shallow hydrothermal vents (White Point, CA) were found at deep-sea hydrothermal vents. These filamentous bacteria grow in dense mats that cover surfaces and potentially provide a significant source of organic carbon where they occur. Vacuolate-attached filaments were collected near vents at the Clam Bed site of the Endeavour Segment of the Juan de Fuca Ridge and from the sed...

  3. The contractile vacuole in Ca2+-regulation in Dictyostelium: its essential function for cAMP-induced Ca2+-influx

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    Schlatterer Christina

    2006-06-01

    Full Text Available Abstract Background cAMP-induced Ca2+-influx in Dictyostelium is controlled by at least two non-mitochondrial Ca2+-stores: acidic stores and the endoplasmic reticulum (ER. The acidic stores may comprise the contractile vacuole network (CV, the endosomal compartment and acidocalcisomes. Here the role of CV in respect to function as a potential Ca2+-store was investigated. Results Dajumin-GFP labeled contractile vacuoles were purified 7-fold by anti-GFP-antibodies in a magnetic field. The purified CV were shown for the first time to accumulate and release Ca2+. Release of Ca2+ was elicited by arachidonic acid or the calmodulin antagonist W7, the latter due to inhibition of the pump. The characteristics of Ca2+-transport and Ca2+-release of CV were compared to similarly purified vesicles of the ER labeled by calnexin-GFP. Since the CV proved to be a highly efficient Ca2+-compartment we wanted to know whether or not it takes part in cAMP-induced Ca2+-influx. We made use of the LvsA--mutant expected to display reduced Ca2+-transport due to loss of calmodulin. We found a severe reduction of cAMP-induced Ca2+-influx into whole cells. Conclusion The contractile vacuoles in Dictyostelium represent a highly efficient acidic Ca2+-store that is required for cAMP-induced Ca2+-influx.

  4. IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

    Science.gov (United States)

    Naujoks, Jan; Kunze, Mareike; Kempa, Stefan; Peter, Andrea; Mollenkopf, Hans-Joachim; Dorhoi, Anca; Kershaw, Olivia; Gruber, Achim D.; Sander, Leif E.; Witzenrath, Martin; Herold, Susanne; Nerlich, Andreas; Hocke, Andreas C.; van Driel, Ian; Suttorp, Norbert; Bedoui, Sammy; Hilbi, Hubert; Trost, Matthias; Opitz, Bastian

    2016-01-01

    Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria. PMID:26829557

  5. Regulation of mucous acinar exocrine secretion with age.

    Science.gov (United States)

    Culp, D J; Richardson, L A

    1996-01-01

    Denny and co-workers (Navazesh et al., 1992) recently reported decreased concentrations of MG1 and MG2 mucins in resting and stimulated whole human saliva with age. The current study was therefore conducted to examine whether there is a corresponding attenuation with age in stimulus secretion coupling regulating mucous cell exocrine secretion. We utilized an in vitro model system, isolated rat sublingual acini, to evaluate the regulation of mucous cell exocrine secretion. Rat sublingual glands are similar to human sublingual and minor mucous glands, both histologically and in terms of their pattern of innervation, which is predominantly parasympathetic. Mucin secretion is thus activated primarily by muscarinic cholinergic agonist and to a lesser extent by vasoactive intestinal peptide (VIP), which is co-localized with acetylcholine in parasympathetic nerve terminals. We isolated sublingual mucous acini from five-month-old and 24-month-old rats and compared the concentration responses for mucin secretion induced by VIP and the muscarinic agonist, arecaidine propargyl ester (APE). Concentration-response curves for VIP were nearly identical for mucous acini from the five-month-old and 24-month-old animals. Values for basal secretion, maximal secretion, and EC50 (approximately equal to 200 nmol/L VIP) were statistically equivalent between both age groups. Concentration-response curves for APE were also very similar between age groups, with no statistically significant difference in basal secretion or EC50 values (approximately equal to 50 nmol/L APE). Maximal secretion was slightly less but statistically different for 24-month-old vs. five-month-old animals, 158% vs. 169% above basal secretion, respectively. Collectively, we found no substantial age-related changes in the secretory responsiveness of salivary mucous cells.

  6. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

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    Cristian A. Carrión

    2014-11-01

    Full Text Available Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs, characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves.

  7. A RabGAP protein and BEACH Family proteins regulate contractile vacuole formation and activity and chemotaxis in Dictyostelium

    OpenAIRE

    Du, Fei

    2007-01-01

    The contractile vacuole (CV) system is the osmoregulatory organelle of free-living amoebae and protozoa. I present data showing that the RabGAP RabGAP1 acts as a switch for discharging the CVs into the extracellular medium in Dictyostelium. rabgap1 null (rabgap1⁻) cells have highly enlarged CVs whose structure and activity are aberrant. In rabgap1- cells, the dynamic fusion of the CV with the plasma membrane is absent and the discharge of CV content is inefficient. RabGAP1 localizes to the CV...

  8. Legionella pneumophila exploits PI(4P to anchor secreted effector proteins to the replicative vacuole.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The causative agent of Legionnaires' disease, Legionella pneumophila, employs the intracellular multiplication (Icm/defective organelle trafficking (Dot type IV secretion system (T4SS to upregulate phagocytosis and to establish a replicative vacuole in amoebae and macrophages. Legionella-containing vacuoles (LCVs do not fuse with endosomes but recruit early secretory vesicles. Here we analyze the role of host cell phosphoinositide (PI metabolism during uptake and intracellular replication of L. pneumophila. Genetic and pharmacological evidence suggests that class I phosphatidylinositol(3 kinases (PI3Ks are dispensable for phagocytosis of wild-type L. pneumophila but inhibit intracellular replication of the bacteria and participate in the modulation of the LCV. Uptake and degradation of an icmT mutant strain lacking a functional Icm/Dot transporter was promoted by PI3Ks. We identified Icm/Dot-secreted proteins which specifically bind to phosphatidylinositol(4 phosphate (PI(4P in vitro and preferentially localize to LCVs in the absence of functional PI3Ks. PI(4P was found to be present on LCVs using as a probe either an antibody against PI(4P or the PH domain of the PI(4P-binding protein FAPP1 (phosphatidylinositol(4 phosphate adaptor protein-1. Moreover, the presence of PI(4P on LCVs required a functional Icm/Dot T4SS. Our results indicate that L. pneumophila modulates host cell PI metabolism and exploits the Golgi lipid second messenger PI(4P to anchor secreted effector proteins to the LCV.

  9. Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

    Science.gov (United States)

    Srinivasan, Padmanabhan; Thrower, Edwin C; Gorelick, Fred S; Said, Hamid M

    2016-05-15

    Thiamin is essential for normal metabolism in pancreatic acinar cells (PAC) and is obtained from their microenvironment through specific plasma-membrane transporters, converted to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria through the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). TPP is essential for normal mitochondrial function. We examined the effect of long-term/chronic exposure of PAC in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type or transgenic mice carrying the SLC25A19 promoter) of the cigarette smoke toxin, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the MTPP uptake process. Our in vitro and in vivo findings demonstrate that NNK negatively affects MTPP uptake and reduced expression of MTPPT protein, MTPPT mRNA, and heterogenous nuclear RNA, as well as SLC25A19 promoter activity. The effect of NNK on Slc25a19 transcription was neither mediated by changes in expression of transcriptional factor NFY-1 (known to drive SLC25A19 transcription), nor due to changes in methylation profile of the Slc25a19 promoter. Rather, it appears to be due to changes in histone modifications that involve significant decreases in histone H3K4-trimethylation and H3K9-acetylation (activation markers). The effect of NNK on MTPPT function is mediated through the nonneuronal α7-nicotinic acetylcholine receptor (α7-nAChR), as indicated by both in vitro (using the nAChR antagonist mecamylamine) and in vivo (using an α7-nAchR(-/-) mouse model) studies. These findings demonstrate that chronic exposure of PAC to NNK negatively impacts PAC MTPP uptake. This effect appears to be exerted at the level of Slc25a19 transcription, involve epigenetic mechanism(s), and is mediated through the α7-nAchR. PMID:26999808

  10. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

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    Lara J. Kohler

    2016-07-01

    Full Text Available Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella-containing vacuole (CCV requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection.

  11. Maize cytokinin dehydrogenase isozymes are localized predominantly to the vacuoles.

    Science.gov (United States)

    Zalabák, David; Johnová, Patricie; Plíhal, Ondřej; Šenková, Karolina; Šamajová, Olga; Jiskrová, Eva; Novák, Ondřej; Jackson, David; Mohanty, Amitabh; Galuszka, Petr

    2016-07-01

    The maize genome encompasses 13 genes encoding for cytokinin dehydrogenase isozymes (CKXs). These enzymes are responsible for irreversible degradation of cytokinin plant hormones and thus, contribute regulating their levels. Here, we focus on the unique aspect of CKXs: their diverse subcellular distribution, important in regulating cytokinin homeostasis. Maize CKXs were tagged with green fluorescent protein (GFP) and transiently expressed in maize protoplasts. Most of the isoforms, namely ZmCKX1, ZmCKX2, ZmCKX4a, ZmCKX5, ZmCKX6, ZmCKX8, ZmCKX9, and ZmCKX12, were associated with endoplasmic reticulum (ER) several hours after transformation. GFP-fused CKXs were observed to accumulate in putative prevacuolar compartments. To gain more information about the spatiotemporal localization of the above isoforms, we prepared stable expression lines of all ZmCKX-GFP fusions in Arabidopsis thaliana Ler suspension culture. All the ER-associated isoforms except ZmCKX1 and ZmCKX9 were found to be targeted primarily to vacuoles, suggesting that ER-localization is a transition point in the intracellular secretory pathway and vacuoles serve as these isoforms' final destination. ZmCKX9 showed an ER-like localization pattern similar to those observed in the transient maize assay. Apoplastic localization of ZmCKX1 was further confirmed and ZmCKX10 showed cytosolic/nuclear localization due to the absence of the signal peptide sequence as previously reported. Additionally, we prepared GFP-fused N-terminal signal deletion mutants of ZmCKX2 and ZmCKX9 and clearly demonstrated that the localization pattern of these mutant forms was cytosolic/nuclear. This study provides the first complex model for spatiotemporal localization of the key enzymes of the cytokinin degradation/catabolism in monocotyledonous plants. PMID:27031423

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

    OpenAIRE

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

    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 ove...

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

    Science.gov (United States)

    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.

  14. The nature of human sperm head vacuoles: a systematic literature review.

    Science.gov (United States)

    Boitrelle, Florence; Guthauser, Bruno; Alter, Laura; Bailly, Marc; Wainer, Robert; Vialard, François; Albert, Martine; Selva, Jacqueline

    2013-01-01

    Motile sperm organelle morphology examination (MSOME) involves the use of differential interference contrast microscopy (also called Nomarski contrast) at high magnification (at least 6300x) to improve the observation of live human spermatozoa. In fact, this technique evidences sperm head vacuoles that are not necessarily seen at lower magnifications - particularly if the vacuoles are small (i.e. occupying nature. In an attempt to clarify this debate, we performed a systematic literature review in accordance with the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms "MSOME", "human sperm vacuoles", "high-magnification, sperm". Out of 180 search results, 21 relevant English-language publications on the nature of human sperm head vacuoles were finally selected and reviewed. Our review of the literature prompted us to conclude that sperm-head vacuoles are nuclear in nature and are related to chromatin condensation failure and (in some cases) sperm DNA damage.

  15. Food vacuole associated enolase in plasmodium undergoes multiple post-translational modifications: evidence for atypical ubiquitination.

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    Saudamini Shevade

    Full Text Available Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS, definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1 and the C-terminal G76 of the second (Ub2. Ub2 and third ubiquitin (Ub3 were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.

  16. Human GBP1 does not localize to pathogen vacuoles but restricts Toxoplasma gondii.

    Science.gov (United States)

    Johnston, Ashleigh C; Piro, Anthony; Clough, Barbara; Siew, Malvin; Virreira Winter, Sebastian; Coers, Jörn; Frickel, Eva-Maria

    2016-08-01

    Guanylate binding proteins (GBPs) are a family of large interferon-inducible GTPases that are transcriptionally upregulated upon infection with intracellular pathogens. Murine GBPs (mGBPs) including mGBP1 and 2 localize to and disrupt pathogen-containing vacuoles (PVs) resulting in the cell-autonomous clearing or innate immune detection of PV-resident pathogens. Human GBPs (hGBPs) are known to exert antiviral host defense and activate the NLRP3 inflammasome, but it is unclear whether hGBPs can directly recognize and control intravacuolar pathogens. Here, we report that endogenous or ectopically expressed hGBP1 fails to associate with PVs formed in human cells by the bacterial pathogens Chlamydia trachomatis or Salmonella typhimurium or the protozoan pathogen Toxoplasma gondii. While we find that hGBP1 expression has no discernible effect on intracellular replication of C. trachomatis and S. typhimurium, we observed enhanced early Toxoplasma replication in CRISPR hGBP1-deleted human epithelial cells. We thus identified a novel role for hGBP1 in cell-autonomous immunity that is independent of PV translocation, as observed for mGBPs. This study highlights fundamental differences between human and murine GBPs and underlines the need to study the functions of GBPs at cellular locations away from PVs. PMID:26874079

  17. Maize ZmVPP5 is a truncated Vacuole Hþ-PPase that confers hypersensitivity to salt stress

    Institute of Scientific and Technical Information of China (English)

    Xiaoliang Sun; Weiwei Qi; Yihong Yue; Huiling Ling; Gang Wang; Rentao Song

    2016-01-01

    In plants, Vacuole Hþ-PPases (VPPs) are important proton pumps and encoded by multiple genes. In addition to full-length VPPs, several truncated forms are expressed, but their biological functions are unknown. In this study, we functionally characterized maize vacuole Hþ-PPase 5 (ZmVPP5), a truncated VPP in the maize genome. Although ZmVPP5 shares high sequence similarity with ZmVPP1, ZmVPP5 lacks the complete structure of the conserved proton transport and the inorganic pyrophosphatase-related domain. Phylogenetic analysis suggests that ZmVPP5 might be derived from an incomplete gene duplication event. ZmVPP5 is expressed in multiple tissues, and ZmVPP5 was detected in the plasma membrane, vacuole membrane and nuclei of maize cells. The overexpression of ZmVPP5 in yeast cells caused a hypersensitivity to salt stress. Transgenic maize lines with overexpressed ZmVPP5 also exhibited the salt hypersensitivity phenotype. A yeast two-hybrid analysis identified the ZmBag6-like protein as a putative ZmVPP5-interacting protein. The results of bimolecular luminescence complementation (BiLC) assay suggest an interaction between ZmBag6-like protein and ZmVPP5 in vivo. Overall, this study suggests that ZmVPP5 might act as a VPP antagonist and participate in the cellular response to salt stress. Our study of ZmVPP5 has expanded the understanding of the origin and functions of truncated forms of plant VPPs.

  18. The C Isoform of Dictyostelium Tetraspanins Localizes to the Contractile Vacuole and Contributes to Resistance against Osmotic Stress.

    Science.gov (United States)

    Albers, Tineke; Maniak, Markus; Beitz, Eric; von Bülow, Julia

    2016-01-01

    Tetraspanins (Tsps) are membrane proteins that are widely expressed in eukaryotic organisms. Only recently, Tsps have started to acquire relevance as potential new drug targets as they contribute, via protein-protein interactions, to numerous pathophysiological processes including infectious diseases and cancer. However, due to a high number of isoforms and functional redundancy, knowledge on specific functions of most Tsps is still scarce. We set out to characterize five previously annotated Tsps, TspA-E, from Dictyostelium discoideum, a model for studying proteins that have human orthologues. Using reverse transcriptase PCRs, we found mRNAs for TspA-E in the multicellular slug stage, whereas vegetative cells expressed only TspA, TspC and, to a lesser extent, TspD. We raised antibodies against TspA, TspC and TspD and detected endogenous TspA, as well as heterologously expressed TspA and TspC by Western blot. N-deglycosylation assays and mutational analyses showed glycosylation of TspA and TspC in vivo. GFP-tagged Tsps co-localized with the proton pump on the contractile vacuole network. Deletion strains of TspC and TspD exibited unaltered growth, adhesion, random motility and development. Yet, tspC- cells showed a defect in coping with hypo-osmotic stress, due to accumulation of contractile vacuoles, but heterologous expression of TspC rescued their phenotype. In conclusion, our data fill a gap in Dictyostelium research and open up the possibility that Tsps in contractile vacuoles of e.g. Trypanosoma may one day constitute a valuable drug target for treating sleeping sickness, one of the most threatening tropical diseases. PMID:27597994

  19. Motile sperm organelle morphology examination (MSOME) and sperm head vacuoles: state of the art in 2013.

    Science.gov (United States)

    Perdrix, Anne; Rives, Nathalie

    2013-01-01

    BACKGROUND Approximately 10 years after the first publication introducing the motile sperm organelle morphology examination (MSOME), many questions remained about sperm vacuoles: frequency, size, localization, mode of occurrence, biological significance and impact on male fertility potential. Many studies have tried to characterize sperm vacuoles, to determine the sperm abnormalities possibly associated with vacuoles, to test the diagnostic value of MSOME for male infertility or to question the benefits of intracytoplasmic morphologically selected sperm injection (IMSI). METHODS We searched PubMed for articles in the English language published in 2001-2012 regarding human sperm head vacuoles, MSOME and IMSI. RESULTS A bibliographic analysis revealed consensus for the following findings: (i) sperm vacuoles appeared frequently, often multiple and preferentially anterior; (ii) sperm vacuoles and sperm chromatin immaturity have been associated, particularly in the case of large vacuoles; (iii) teratozoospermia was a preferred indication of MSOME and IMSI. CONCLUSION The high-magnification system appears to be a powerful method to improve our understanding of human spermatozoa. However, its clinical use remains unclear in the fields of male infertility diagnosis and assisted reproduction techniques (ARTs). PMID:23825157

  20. MTMR4 Is Required for the Stability of the Salmonella-Containing Vacuole.

    Science.gov (United States)

    Teo, Wei X; Kerr, Markus C; Teasdale, Rohan D

    2016-01-01

    The intracellular pathogen Salmonella enterica servovar Typhimurium (S.typhimurium) modulates the host cell's phosphoinositide (PI) metabolism to establish its intracellular replicative niche, the Salmonella-containing vacuole (SCV). Upon invasion, phosphoinositide 3-phosphate (PI(3)P) and other early endosomal markers are rapidly recruited to and remain associated with the SCV throughout its early maturation. While the phosphoinositide 3-phosphatase myotubularin 4 (MTMR4) has an established role in regulating autophagy and cellular PI(3)P-content, two processes associated with the intracellular survival of S. typhimurium, a direct role for MTMR4 in Salmonella biology has not been examined. Here we demonstrate that GFP-tagged MTMR4 is recruited to the SCV and infection of cells depleted of endogenous MTMR4 results in a decrease in viable intracellular Salmonella. This reflects a significant increase in the proportion of SCVs with compromised integrity, which targets the compartment for autophagy and consequent bacterial cell death. These findings highlight the importance of PI(3)P regulation to the integrity of the SCV and reveal a novel role for the myotubularins in bacterial pathogenesis. PMID:27625994

  1. The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

    Directory of Open Access Journals (Sweden)

    Fernando Real

    Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

  2. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

  3. Overexpression of calsequestrin in L6 myoblasts: formation of endoplasmic reticulum subdomains and their evolution into discrete vacuoles where aggregates of the protein are specifically accumulated.

    OpenAIRE

    Gatti, G.; Podini, P; Meldolesi, J

    1997-01-01

    Calsequestrin (CSQ), the major low-affinity Ca(2+)-binding glycoprotein of striated muscle fibers, is concentrated to yield aggregates that occupy the lumen of the terminal cisternae of the sarcoplasmic reticulum (SR). When infected or transfected into L6 myoblast, the protein is also concentrated, however, in dense vacuoles apparently separate from the endoplasmic reticulum (ER). CSQ-rich cells appear otherwise normal; in particular, neither other proteins involved in Ca2+ homeostasis nor ER...

  4. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

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    Lukas Aeberhard

    2015-06-01

    Full Text Available 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.

  5. Solute accumulation differs in the vacuoles and apoplast of ripening grape berries.

    Science.gov (United States)

    Keller, Markus; Shrestha, Pradeep M

    2014-03-01

    Phloem unloading is thought to switch from a symplastic route to an apoplastic route at the beginning of ripening in grape berries and some other fleshy fruits. However, it is unclear whether different solutes accumulate in both the mesocarp vacuoles and the apoplast. We modified a method developed for tomato fruit to extract apoplastic sap from grape berries and measured the changes in apoplastic and vacuolar pH, soluble sugars, organic acids, and potassium in ripening berries of Vitis vinifera 'Merlot' and V. labruscana 'Concord'. Solute accumulation varied by genotype, compartment, and chemical species. The apoplast pH was substantially higher than the vacuolar pH, especially in Merlot (approximately two units). However, the vacuole-apoplast proton gradient declined during ripening and in Merlot, but not in Concord, collapsed entirely at maturity. Hexoses accumulated in both the vacuoles and apoplast but at different rates. Organic acids, especially malate, declined much more in the vacuoles than in the apoplast. Potassium accumulated in the vacuoles and apoplast of Merlot. In Concord, by contrast, potassium increased in the vacuoles but decreased in the apoplast. These results suggest that solutes in the fruit apoplast are tightly regulated and under developmental control. PMID:24310282

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

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    Suto Jun-ichi

    2012-11-01

    Full Text Available Abstract 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

  7. Abscisic acid prevents the coalescence of protein storage vacuoles by upregulating expression of a tonoplast intrinsic protein gene in barley aleurone.

    Science.gov (United States)

    Lee, Sung-eun; Yim, Hui-kyung; Lim, Mi-na; Yoon, In sun; Kim, Jeong hoe; Hwang, Yong-sic

    2015-03-01

    Tonoplast intrinsic proteins (TIPs) are integral membrane proteins that are known to function in plants as aquaporins. Here, we propose another role for TIPs during the fusion of protein storage vacuoles (PSVs) in aleurone cells, a process that is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Studies of the expression of barley (Hordeum vulgare) TIP genes (HvTIP) showed that GA specifically decreased the abundance of HvTIP1;2 and HvTIP3;1 transcripts, while ABA strongly increased expression of HvTIP3;1. Increased or decreased expression of HvTIP3;1 interfered with the hormonal effects on vacuolation in aleurone protoplasts. HvTIP3;1 gain-of-function experiments delayed GA-induced vacuolation, whereas HvTIP3;1 loss-of-function experiments promoted vacuolation in ABA-treated aleurone cells. These results indicate that TIP plays a key role in preventing the coalescence of small PSVs in aleurone cells. Hormonal regulation of the HvTIP3;1 promoter is similar to the regulation of the endogenous gene, indicating that induction of the transcription of HvTIP3;1 by ABA is a critical factor in the prevention of PSV coalescence in response to ABA. Promoter analysis using deletions and site-directed mutagenesis of sequences identified three cis-acting elements that are responsible for ABA responsiveness in the HvTIP3;1 promoter. Promoter analysis also showed that ABA responsiveness of the HvTIP3;1 promoter is likely to occur via a unique regulatory system distinct from that involving the ABA-response promoter complexes.

  8. The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

    DEFF Research Database (Denmark)

    Takeda, Kozue; Cabrera, Margarita; Rohde, Jan;

    2008-01-01

    At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate...

  9. Primary alveolar capillary dysplasia (acinar dysplasia) and surfactant protein B deficiency: a clinical, radiological and pathological study

    Energy Technology Data Exchange (ETDEWEB)

    Hugosson, Claes O.; Khoumais, Nuha [King Faisal Specialist Hospital and Research Centre, Department of Radiology MBC 28, Riyadh (Saudi Arabia); Salama, Husam M.; Kattan, Abdul H. [King Faisal Specialist Hospital and Research Centre, Department of Paediatrics, Riyadh (Saudi Arabia); Al-Dayel, Fouad [King Faisal Specialist Hospital and Research Centre, Department of Pathology, Riyadh (Saudi Arabia)

    2005-03-01

    Full-term infants with severe and prolonged respiratory distress represent a diagnostic challenge. Plain radiographic findings may be nonspecific or similar to classic surfactant deficiency disease for infants with surfactant protein B deficiency and acinar dysplasia. Objectives: To describe the similar clinical-radiolgical patterns of two rare neonatal conditions. Six newborn babies with severe respiratory distress at birth demonstrated clinical and radiographically prolonged and progressive diffuse pulmonary opacification. All infants demonstrated hyperinflation of the lungs. The diffuse hazy opacification, which varied from mild (n=3) to moderate (n=3), progressed to severe diffuse opacification preceding death, which occurred at 12-36 days of life. Open lung biopsy confirmed the diagnosis of primary alveolar acinar dysplasia (AD) in four infants and surfactant protein B deficiency (SPBD) in two infants. In full-term babies with unexplained progressive respiratory distress from birth and progress of radiological changes, both AD and SPBD should be considered. (orig.)

  10. Primary alveolar capillary dysplasia (acinar dysplasia) and surfactant protein B deficiency: a clinical, radiological and pathological study

    International Nuclear Information System (INIS)

    Full-term infants with severe and prolonged respiratory distress represent a diagnostic challenge. Plain radiographic findings may be nonspecific or similar to classic surfactant deficiency disease for infants with surfactant protein B deficiency and acinar dysplasia. Objectives: To describe the similar clinical-radiological patterns of two rare neonatal conditions. Six newborn babies with severe respiratory distress at birth demonstrated clinical and radiographically prolonged and progressive diffuse pulmonary opacification. All infants demonstrated hyperinflation of the lungs. The diffuse hazy opacification, which varied from mild (n=3) to moderate (n=3), progressed to severe diffuse opacification preceding death, which occurred at 12-36 days of life. Open lung biopsy confirmed the diagnosis of primary alveolar acinar dysplasia (AD) in four infants and surfactant protein B deficiency (SPBD) in two infants. In full-term babies with unexplained progressive respiratory distress from birth and progress of radiological changes, both AD and SPBD should be considered. (orig.)

  11. Helicobacter pylori γ-glutamyl transpeptidase and vacuolating cytotoxin promote gastric persistence and immune tolerance.

    Science.gov (United States)

    Oertli, Mathias; Noben, Manuel; Engler, Daniela B; Semper, Raphaela P; Reuter, Sebastian; Maxeiner, Joachim; Gerhard, Markus; Taube, Christian; Müller, Anne

    2013-02-19

    Infection with the gastric bacterial pathogen Helicobacter pylori is typically contracted in early childhood and often persists for decades. The immunomodulatory properties of H. pylori that allow it to colonize humans persistently are believed to also account for H. pylori's protective effects against allergic and chronic inflammatory diseases. H. pylori infection efficiently reprograms dendritic cells (DCs) toward a tolerogenic phenotype and induces regulatory T cells (Tregs) with highly suppressive activity in models of allergen-induced asthma. We show here that two H. pylori virulence determinants, the γ-glutamyl transpeptidase GGT and the vacuolating cytotoxin VacA, contribute critically and nonredundantly to H. pylori's tolerizing effects on murine DCs in vitro and in vivo. The tolerance-promoting effects of both factors are independent of their described suppressive activity on T cells. Isogenic H. pylori mutants lacking either GGT or VacA are incapable of preventing LPS-induced DC maturation and fail to drive DC tolerization as assessed by induction of Treg properties in cocultured naive T cells. The Δggt and ΔvacA mutants colonize mice at significantly reduced levels, induce stronger T-helper 1 (Th1) and T-helper 17 (Th17) responses, and/or trigger more severe gastric pathology. Both factors promote the efficient induction of Tregs in vivo, and VacA is required to prevent allergen-induced asthma. The defects of the Δggt mutant in vitro and in vivo are phenocopied by pharmacological inhibition of the transpeptidase activity of GGT in all readouts. In conclusion, our results reveal the molecular players and mechanistic basis for H. pylori-induced immunomodulation, promoting persistent infection and conferring protection against allergic asthma. PMID:23382221

  12. New challenges for the design of high value plant products: stabilization of anthocyanins in plant vacuoles

    Directory of Open Access Journals (Sweden)

    Valentina ePasseri

    2016-02-01

    Full Text Available In the last decade plant biotechnologists and breeders have made several attempt to improve the antioxidant content of plant-derived food. Most efforts concentrated on increasing the synthesis of antioxidants, in particular anthocyanins, by inducing the transcription of genes encoding the synthesizing enzymes. We present here an overview of economically interesting plant species, both food crops and ornamentals, in which anthocyanin content was improved by traditional breeding or transgenesis. Old genetic studies in petunia and more recent biochemical work in brunfelsia, have shown that after synthesis and compartmentalization in the vacuole, anthocyanins need to be stabilized to preserve the color of the plant tissue over time. The final yield of antioxidant molecules is the result of the balance between synthesis and degradation. Therefore the understanding of the mechanism that determine molecule stabilization in the vacuolar lumen is the next step that needs to be taken to further improve the anthocyanin content in food.In several species a phenomenon known as fading is responsible for the disappearance of pigmentation which in some case can be nearly complete. We discuss the present knowledge about the genetic and biochemical factors involved in pigment preservation/destabilization in plant cells.The improvement of our understanding of the fading process will supply new tools for both biotechnological approaches and marker-assisted breeding.

  13. Plasmodium protease ROM1 is important for proper formation of the parasitophorous vacuole.

    Directory of Open Access Journals (Sweden)

    Iset Medina Vera

    2011-09-01

    Full Text Available Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(- parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(- sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(- sporozoites do not differ in the rate of entry into Hepa1-6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(- parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(- parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host.

  14. Vacuolate-attached filaments: highly productive Ridgeia piscesae epibionts at the Juan de Fuca hydrothermal vents.

    Science.gov (United States)

    Kalanetra, Karen M; Nelson, Douglas C

    2010-01-01

    Vacuolate sulfur bacteria with high morphological similarity to vacuolate-attached filaments previously described from shallow hydrothermal vents (White Point, CA) were found at deep-sea hydrothermal vents. These filamentous bacteria grow in dense mats that cover surfaces and potentially provide a significant source of organic carbon where they occur. Vacuolate-attached filaments were collected near vents at the Clam Bed site of the Endeavour Segment of the Juan de Fuca Ridge and from the sediment surface at Escanaba Trough on the Gorda Ridge. A phylogenetic analysis comparing their 16S rRNA gene sequences to those collected from the shallow White Point site showed that all vacuolate-attached filament sequences form a monophyletic group within the vacuolate sulfur-oxidizing bacteria clade in the gamma proteobacteria. Abundance of the attached filaments was quantified over the length of the exterior surface of the tubes of Ridgeia piscesae worms collected from the Clam Bed site at Juan de Fuca yielding a per worm average of 0.070 ± 0.018 cm(3) (n = 4). In agreement with previous results for White Point filaments, anion measurements by ion chromatography showed no detectable internal nitrate concentrations above ambient seawater (n = 9). For one R. piscesae tube worm "bush" at the Easter Island vent site, potential gross epibiont productivity is estimated to be 15 to 45× the net productivity of the worms. PMID:24391244

  15. Characterization of Helicobacter pylori VacA-containing vacuoles (VCVs), VacA intracellular trafficking and interference with calcium signalling in T lymphocytes.

    Science.gov (United States)

    Kern, Beate; Jain, Utkarsh; Utsch, Ciara; Otto, Andreas; Busch, Benjamin; Jiménez-Soto, Luisa; Becher, Dörte; Haas, Rainer

    2015-12-01

    The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases such as gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion-selective channels in the endosome membrane that cause the compartment to swell, but the composition and purpose of the resulting VacA-containing vacuoles (VCVs) are still unknown. VacA exerts influence on the host immune response in various ways, including inhibition of T-cell activation and proliferation and suppression of the host immune response. In this study, for the first time the composition of VCVs from T cells was comprehensively analysed to investigate VCV function. VCVs were successfully isolated via immunomagnetic separation, and the purified vacuoles were analysed by mass spectrometry. We detected a set of 122 VCV-specific proteins implicated among others in immune response, cell death and cellular signalling processes, all of which VacA is known to influence. One of the individual proteins studied further was stromal interaction molecule (STIM1), a calcium sensor residing in the endoplasmic reticulum (ER) that is important in store-operated calcium entry. Live cell imaging microscopy data demonstrated colocalization of VacA with STIM1 in the ER and indicated that VacA may interfere with the movement of STIM1 towards the plasma membrane-localized calcium release activated calcium channel protein ORAI1 in response to Ca(2+) store depletion. Furthermore, VacA inhibited the increase of cytosolic-free Ca(2+) in the Jurkat E6-1 T-cell line and human CD4(+) T cells. The presence of VacA in the ER and its trafficking to the Golgi apparatus was confirmed in HeLa cells, identifying these two cellular compartments as novel VacA target structures.

  16. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated memb

  17. Biopsy follow-up in patients with isolated atypical small acinar proliferation (ASAP in prostate biopsy

    Directory of Open Access Journals (Sweden)

    Luca Leone

    2014-12-01

    Full Text Available The incidence of prostate cancer (PCA was evaluated in 155 patients with isolated Atypical Small Acinar Proliferation (ASAP found on initial prostate biopsy, after a medium-term follow-up (40 months with at least one re-biopsy. Clinical and histological data were analysed. Cancer was detected in 81 of 155 (52.3%. The cancer detection rate was 71.6%, 91.3%, 97.5%, 100% at the 1st re-biopsy, 2nd, 3rd, and 4th rebiopsy respectively. At the uni- and multivariate analyses, prostate volume (≤ 30 cc, transition zone volume (≤ 10 cc, small core length at the initial biopsy (≤ 10 mm and few number of cores at initial biopsy (≤ 8 are predictive of cancer. Furthermore, tumour characteristics on the whole surgical specimens was assessed in 30 men: 13 of 30 (43 % had clinically relevant cancer (volume > 0.5 ml or/and Gleason score ≥ 7, or pT3. Most of relevant cancers were detected in the distal apex, anterior gland and midline. These anatomical sites could be under-sampled at the initial biopsy using the transrectal approach. Our data suggest that follow-up biopsy is recommended in all cases of isolated ASAP detected after biopsy using endfire transrectal probe. The re-biopsy strategy should increase the number of cores (or a saturation biopsy, focusing on area of ASAP in the initial biopsy, but also including the under-sampled areas (anterior gland, distal apex and midline to detect clinically relevant cancers.

  18. Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

    Directory of Open Access Journals (Sweden)

    Peng Liu

    Full Text Available The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD. We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.

  19. Morphology changes in human lung epithelial cells after exposure to diesel exhaust micron sub particles (PM1.0) and pollen allergens

    International Nuclear Information System (INIS)

    In the recent literature there has been an increased interest in the effects of particulate matter on the respiratory tract. The objective of this study was to use an in vitro model of type II lung epithelium (A549) to evaluate the cell ability to take up sub-micron PM1.0 particles (PM1.0), Parietaria officinalis (ALL), and PM1.0 + ALL together. Morphological analysis performed by Transmission Electron Microscope (TEM) showed that PM and ALL interacted with the cell surface, then penetrating into the cytoplasm. Each single treatment was able to point out a specific change in the morphology. The cells treated appear healthy and not apoptotic. The main effect was the increase of: multilamellar bodies, lysosomal enzymes, microvilli, and presence of vesicle/vacuoles containing particles. These observations demonstrate morphological and functional alterations related to the PM1.0 and P. officinalis and confirm the induction of the inflammatory response in lung cells exposed to the inhalable particles. - Highlights: ► Cell ability to take up PM1.0 particles, Parietaria officinalis (ALL), PM1.0 + ALL. ► The cells treated appear healthy and not apoptotic. ► Each single treatment was able to point out a specific change in the morphology. ► Increase of multilamellar bodies lysosomal enzymes microvilli vesicle with particles. ► Induction of inflammatory response in lung cells exposed to the inhalable particles. - The urban environment with the combination of inhalable air pollution and particulate can damage the acinar lung units and activate cells of the immune system.

  20. Rimmed vacuoles and the added value of SMI-31 staining in diagnosing sporadic inclusion body myositis.

    Science.gov (United States)

    van der Meulen, M F; Hoogendijk, J E; Moons, K G; Veldman, H; Badrising, U A; Wokke, J H

    2001-07-01

    Problems in diagnosing sporadic inclusion body myositis may arise if all clinical features fit a diagnosis of polymyositis, but the muscle biopsy shows some rimmed vacuoles. Recently, immunohistochemistry with an antibody directed against phosphorylated neurofilament (SMI-31) has been advocated as a diagnostic test for sporadic inclusion body myositis. The aims of the present study were to define a quantitative criterion to differentiate sporadic inclusion body myositis from polymyositis based on the detection of rimmed vacuoles in the haematoxylin-eosin staining and to evaluate the additional diagnostic value of the SMI-31 staining. Based on clinical criteria and creatine kinase levels in patients with endomysial infiltrates, 18 patients complied with the diagnosis of sporadic inclusion body myositis, and 17 with the diagnosis of polymyositis. A blinded observer counted the abnormal fibres in haematoxylin-eosin-stained sections and in SMI-31-stained sections. The optimal cut-off in the haematoxylin-eosin test was 0.3% vacuolated fibres. Adding the SMI-31 staining significantly increased the positive predictive value from 87 to 100%, but increased the negative predictive value only to small extent. We conclude that (1) patients with clinical and laboratory features of polymyositis, including response to treatment, may show rimmed vacuoles in their muscle biopsy and that (2) adding the SMI-31 stain can be helpful in differentiating patients who respond to treatment from patients who do not.

  1. Oculopharyngeal Weakness, Hypophrenia, Deafness, and Impaired Vision: A Novel Autosomal Dominant Myopathy with Rimmed Vacuoles

    Science.gov (United States)

    Chen, Ting; Lu, Xiang-Hui; Wang, Hui-Fang; Ban, Rui; Liu, Hua-Xu; Shi, Qiang; Wang, Qian; Yin, Xi; Pu, Chuan-Qiang

    2016-01-01

    Background: Myopathies with rimmed vacuoles are a heterogeneous group of muscle disorders with progressive muscle weakness and varied clinical manifestations but similar features in muscle biopsies. Here, we describe a novel autosomal dominant myopathy with rimmed vacuoles in a large family with 11 patients of three generations affected. Methods: A clinical study including family history, obstetric, pediatric, and development history was recorded. Clinical examinations including physical examination, electromyography (EMG), serum creatine kinase (CK), bone X-rays, and brain magnetic resonance imaging (MRI) were performed in this family. Open muscle biopsies were performed on the proband and his mother. To find the causative gene, the whole-exome sequencing was carried out. Results: Disease onset was from adolescence to adulthood, but the affected patients of the third generation presented an earlier onset and more severe clinical manifestations than the older generations. Clinical features were characterized as dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision. However, not every patient manifested all symptoms. Serum CK was mildly elevated and EMG indicated a myopathic pattern. Brain MRI showed cerebellum and brain stem mildly atrophy. Rimmed vacuoles and inclusion bodies were observed in muscle biopsy. The whole-exome sequencing was performed, but the causative gene has not been found. Conclusions: We reported a novel autosomal dominant myopathy with rimmed vacuoles characterized by dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision, but the causative gene has not been found and needs further study. PMID:27453229

  2. Polar transmembrane domains target proteins to the interior of the yeast vacuole

    NARCIS (Netherlands)

    Reggiori, F; Black, M W; Pelham, H R; Reggiori, Fulvio

    2000-01-01

    Membrane proteins transported to the yeast vacuole can have two fates. Some reach the outer vacuolar membrane, whereas others enter internal vesicles, which form in late endosomes, and are ultimately degraded. The vacuolar SNAREs Nyv1p and Vam3p avoid this fate by using the AP-3-dependent pathway, w

  3. Retromer and the dynamin Vps1 cooperate in the retrieval of transmembrane proteins from vacuoles

    NARCIS (Netherlands)

    Arlt, Henning; Reggiori, Fulvio; Ungermann, Christian

    2015-01-01

    Endosomes are dynamic organelles that need to combine the ability to successfully deliver proteins and lipids to the lysosome-like vacuole, and recycle others to the Golgi or the plasma membrane. We now show that retromer, which is implicated in retrieval of proteins from endosomes to the Golgi or t

  4. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki;

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

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

    Science.gov (United States)

    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. PMID:27330712

  6. Vacuolating cytotoxin A (VacA) - A multi-talented pore-forming toxin from Helicobacter pylori.

    Science.gov (United States)

    Junaid, Muhammad; Linn, Aung Khine; Javadi, Mohammad Bagher; Al-Gubare, Sarbast; Ali, Niaz; Katzenmeier, Gerd

    2016-08-01

    Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori. Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori. Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation. This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis. PMID:27105670

  7. MoVam7, a conserved SNARE involved in vacuole assembly, is required for growth, endocytosis, ROS accumulation, and pathogenesis of Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Xianying Dou

    Full Text Available 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.

  8. 蓝藻伪空胞的测定及其前处理方法研究%Measurement of Gas Vacuole in Cyanobacteria and Its Pretreatment Method

    Institute of Scientific and Technical Information of China (English)

    杨波; 储昭升; 潘纲

    2011-01-01

    [Objective] The aim was to measure gas vacuole in cyanobacteria and discuss its pretreatment method. [ Method] The capillary pressure method to determine gas vacuole in cyanobacteria was modified firstly,and then the lower detection limit and precision of modified apparatus were tested,finally the effects of two concentration methods and preservation methods on cell number and gas vacuole of cyanobacteria were studied. [Result] The lower detection limit and relative standard deviation of modified apparatus to measure gas vacuole in three kinds of cyanobacteria were 0.001 8 μl/ml and 1% .respectively. Unicellular Microcystis couldn't be concentrated effectively by filtration or centrifugation method,and the lost rate reached 50%. However,the colony of Microcystis and filamentous Planktothrix mougeotii could be concentrated by centrifugation and filtration method, respectively, with lower loss rate. After preserved by direct refrigeration and adding Lugol' s iodine solution for 7 d, there was no obvious change in cell concentration and gas vacuole content per cell,and the loss of direct refrigeration was small for gas vacuole,while the preservation of natura water samples should add Lugol's iodine solution. [Conclusion] The study could provide theoretical foundation for the researches on buoyancy regulation mechanism and of cyanobacteria.%[目的]探讨蓝藻伪空胞的测定及其前处理方法.[方法]改进了测定伪空胞的毛细管压力法,对改进后的装置进行了检测限和精密度测试,并研究了2种浓缩方法和2种保存方法对3种蓝藻细胞数量和伪空胞含量的影响.[结果]改进后的蓝藻伪空胞测定装置其检测限达到0.001 8μl/ml藻液,同一样品测定结果相对标准偏差小于1%.采用过滤和离心2种浓缩方法时,分散单细胞的微囊藻细胞损失都大于50%,其中小群体的微囊藻采用离心的方法细胞损失较小,而丝状的颤藻采用过滤的方法损失较小;这2种方

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

    OpenAIRE

    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 fro...

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

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Amrita; Nandi, Kamal K. [Department of Mathematics, University of North Bengal, Raja Rammohunpur, Siliguri 734 013 (India); Garipova, Guzel M. [Department of Theoretical Physics, Sterlitamak State Pedagogical Academy, 49, Lenin Street, Sterlitamak 453103 (Russian Federation); Laserra, Ettore [DMI, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno (Italy); Bhadra, Arunava, E-mail: amrita852003@yahoo.co.in, E-mail: goldberg144@gmail.com, E-mail: elaserra@unisa.it, E-mail: aru_bhadra@yahoo.com, E-mail: kamalnandi1952@yahoo.co.in [High Energy and Cosmic Ray Research Center, University of North Bengal, Raja Rammohunpur, Siliguri 734 013 (India)

    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. 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.

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

    Science.gov (United States)

    Bhattacharya, Amrita; Garipova, Guzel M.; Laserra, Ettore; Bhadra, Arunava; Nandi, Kamal K.

    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 M3 and confirm the light bending obtained previously in their vacuole model by Ishak et al. (2008). We also obtain another local coupling term -5πM2Λ/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. 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.

  12. Delivery of a secreted soluble protein to the vacuole via a membrane anchor

    Energy Technology Data Exchange (ETDEWEB)

    Barrieu, F.; Chrispeels, M.J.

    1999-08-01

    To further understand how membrane proteins are sorted in the secretory system, the authors devised a strategy that involves the expression of a membrane-anchored yeast invertase in transgenic plants. The construct consisted of a signal peptide followed by the coding region of yeast invertase and the transmembrane domain and cytoplasmic tail of calnexin. The substitution of a lysine near the C terminus of calnexin with a glutamic acid residue ensured progression through the secretory system rather than retention in or return to the endoplasmic reticulum. In the transformed plants, invertase activity and a 70-kD cross-reacting protein were found in the vacuoles. This yeast invertase had plant-specific complex glycans, indicating that transport to the vacuole was mediated by the Golgi apparatus. The microsomal fraction contained a membrane-anchored 90-kD cross-reacting polypeptide, but was devoid of invertase activity. Their results indicate that this membrane-anchored protein proceeds in the secretory system beyond the point where soluble proteins are sorted for secretion, and is detached from its membrane anchor either just before or just after delivery to the vacuole.

  13. Saccharomyces cerevisiae Is Dependent on Vesicular Traffic between the Golgi Apparatus and the Vacuole When Inositolphosphorylceramide Synthase Aur1 Is Inactivated.

    Science.gov (United States)

    Voynova, Natalia S; Roubaty, Carole; Vazquez, Hector M; Mallela, Shamroop K; Ejsing, Christer S; Conzelmann, Andreas

    2015-12-01

    Inositolphosphorylceramide (IPC) and its mannosylated derivatives are the only complex sphingolipids of yeast. Their synthesis can be reduced by aureobasidin A (AbA), which specifically inhibits the IPC synthase Aur1. AbA reportedly, by diminishing IPC levels, causes endoplasmic reticulum (ER) stress, an increase in cytosolic calcium, reactive oxygen production, and mitochondrial damage leading to apoptosis. We found that when Aur1 is gradually depleted by transcriptional downregulation, the accumulation of ceramides becomes a major hindrance to cell survival. Overexpression of the alkaline ceramidase YPC1 rescues cells under this condition. We established hydroxylated C26 fatty acids as a reliable hallmark of ceramide hydrolysis. Such hydrolysis occurs only when YPC1 is overexpressed. In contrast, overexpression of YPC1 has no beneficial effect when Aur1 is acutely repressed by AbA. A high-throughput genetic screen revealed that vesicle-mediated transport between Golgi apparatus, endosomes, and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and quinacrine uptake into vacuoles shows that AbA activates vacuolar acidification. The antioxidant N-acetylcysteine does not improve cell growth on AbA, indicating that reactive oxygen radicals induced by AbA play a minor role in its toxicity. AbA strongly induces the cell wall integrity pathway, but osmotic support does not improve the viability of wild-type cells on AbA. Altogether, the data support and refine current models of AbA-mediated cell death and add vacuolar protein transport and acidification as novel critical elements of stress resistance.

  14. The degradation of potato virus M (PVM particles in plant cells

    Directory of Open Access Journals (Sweden)

    Anna Rudzińska-Langwald

    2014-02-01

    Full Text Available Degradation of potato virus M particles was observed in the cells of Solanum tuberosum, Solanum rostratum, Lycopersicon esculentum and Lycopersicon chilense plants infected with this virus. PVM particles found in the cytoplasm of infected parenchyma cells grouped together in the form of inclusions, often found near the tonoplast. The ends of the virus particles and the tonoplast came into close contact. Cytoplasmic protrusions containing PVM particles, reaching into vacuoles were formed in those places. In addition to a large central vacuole, small vacuoles were observed in cells containing PVM particles. Various stages of degradation of cytoplasmic protrusions were observed both in the large and small vacuoles.

  15. Legionella pneumophila Type IV Effectors YlfA and YlfB Are SNARE-Like Proteins that Form Homo- and Heteromeric Complexes and Enhance the Efficiency of Vacuole Remodeling.

    Directory of Open Access Journals (Sweden)

    Eva M Campodonico

    Full Text Available Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires' disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment.

  16. Legionella pneumophila Type IV Effectors YlfA and YlfB Are SNARE-Like Proteins that Form Homo- and Heteromeric Complexes and Enhance the Efficiency of Vacuole Remodeling

    Science.gov (United States)

    Campodonico, Eva M.; Roy, Craig R.; Ninio, Shira

    2016-01-01

    Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires’ disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER)-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment. PMID:27459495

  17. Legionella pneumophila Type IV Effectors YlfA and YlfB Are SNARE-Like Proteins that Form Homo- and Heteromeric Complexes and Enhance the Efficiency of Vacuole Remodeling.

    Science.gov (United States)

    Campodonico, Eva M; Roy, Craig R; Ninio, Shira

    2016-01-01

    Legionella pneumophila is a Gram-negative bacterium that can colonize both freshwater protozoa and human alveolar macrophages, the latter infection resulting in Legionnaires' disease. The intracellular lifecycle of L. pneumophila requires extensive manipulation of its host cell, which is carried out by effector proteins that are translocated into the host cell through the Dot/Icm type IV secretion system. This study focuses on a pair of highly similar type IV substrates called YlfA/LegC7 and YlfB/LegC2 that were initially identified in a screen for proteins that cause growth inhibition in yeast. Analysis of truncation mutants revealed that the hydrophobic residues in the Ylf amino termini were required for localization of each protein to the membranes of host cells. Central and carboxy terminal coiled coil domains were found to mediate binding of YlfA and YlfB to themselves and to each other. In vivo, a ΔylfA ΔylfB double mutant strain of L. pneumophila was shown to be defective in establishing a vacuole that supports bacterial replication. This phenotype was subsequently correlated with a decrease in the association of endoplasmic reticulum (ER)-derived vesicles with vacuoles containing ΔylfA ΔylfB mutant bacteria. These data suggest that the Ylf proteins are membrane-associated effectors that enhance remodeling of the L. pneumophila -containing vacuole by promoting association and possibly fusion of ER-derived membrane vesicles with the bacterial compartment. PMID:27459495

  18. Matrigel improves functional properties of primary human salivary gland cells.

    Science.gov (United States)

    Maria, Ola M; Zeitouni, Anthony; Gologan, Olga; Tran, Simon D

    2011-05-01

    Currently, there is no effective treatment available to patients with irreversible loss of functional salivary acini caused by Sjogren's syndrome or after radiotherapy for head and neck cancer. A tissue-engineered artificial salivary gland would help these patients. The graft cells for this device must establish tight junctions in addition to being of fluid-secretory nature. This study analyzed a graft source from human salivary glands (huSG) cultured on Matrigel. Cells were obtained from parotid and submandibular glands, expanded in vitro, and then plated on either Matrigel-coated (2 mg/mL) or uncoated culture dish. Immunohistochemistry, transmission electron microscopy, quantitative real-time-polymerase chain reaction, Western blot, and transepithelial electrical resistance were employed. On Matrigel, huSG cells adopted an acinar phenotype by forming three-dimensional acinar-like units (within 24 h of plating) as well as a monolayer of cells. On uncoated surfaces (plastic), huSG cells only formed monolayers of ductal cells. Both types of culture conditions allowed huSG cells to express tight junction proteins (claudin-1, -2, -3, -4; occludin; JAM-A; and ZO-1) and adequate transepithelial electrical resistance. Importantly, 99% of huSG cells on Matrigel expressed α-amylase and the water channel protein Aquaporin-5, as compared to cells on plastic. Transmission electron microscopy confirmed an acinar phenotype with many secretory granules. Matrigel increased the secretion of α-amylase two to five folds into the media, downregulated certain salivary genes, and regulated the translation of acinar proteins. This three-dimensional in vitro serum-free cell culture method allows the organization and differentiation of huSG cells into salivary cells with an acinar phenotype.

  19. Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

    Indian Academy of Sciences (India)

    Katherine Maringer; Azure Yarbrough; Sunder Sims-Lucas; Entsar Saheb; Sanaa Jawed; John Bush

    2016-06-01

    Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

  20. The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2015-12-01

    Full Text Available Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L. was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid and 2.4-D (phenoxyacetic herbicide, characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide and diuron (urea herbicide also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides.

  1. Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.

    Science.gov (United States)

    Finsel, Ivo; Hilbi, Hubert

    2015-07-01

    Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well-characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen-host interactions, but also shed light on novel biological mechanisms.

  2. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    Science.gov (United States)

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  3. The Vacuole Model Revisited: New Repulsive Terms in the Second Order Deflection of Light

    CERN Document Server

    Bhattacharya, Amrita; Potapov, Alexander A; Bhadra, Arunava; Nandi, Kamal K

    2010-01-01

    We calculate the light deflection angle in the Schwarzschild-de Sitter vacuole truly to second order. The derived formulas reveal several new repulsion terms due to the cosmological constant $lambda$, including a modification of the term derived by Ishak et al. (2008). The analysis here also includes the effect of a conformal parameter $gamma$ on light deflection. Much depends on the sign and exact value of $gamma$. Their impact on deflection is addressed. Our deflection calculations naturally reveal an upper limit $lambda$ less than 10E-51. Various deflection components are tabulated at the end.

  4. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

    Science.gov (United States)

    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. PMID:26637549

  5. LegC3, an effector protein from Legionella pneumophila, inhibits homotypic yeast vacuole fusion in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Terry L Bennett

    Full Text Available 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.

  6. Multinodular and vacuolating neuronal tumor affecting amygdala and hippocampus: A quasi-tumor?

    Science.gov (United States)

    Yamaguchi, Maki; Komori, Takashi; Nakata, Yasuhiro; Yagishita, Akira; Morino, Michiharu; Isozaki, Eiji

    2016-01-01

    Multinodular and vacuolating neuronal tumors (MVNT) have been referred to as distinctive neuronal tumors whose characteristic features include multiple nodules localized in the subcortical white matter. MVNT are composed of vacuolating dysplastic neurons reactive to HuC/HuD. A significant overexpression of alpha-internexin (INA) limited to the stroma of nodules was reported in one tumor. Since genetic analyses have failed to demonstrate any consistent alterations, the nosological position as well as the nature of MVNT, namely, neoplastic or dysplastic, remains unclear. We herein present another example of MVNT involving the amygdala and anterior hippocampus in a 41-year-old man. In addition to the nodular lesions described earlier, we found INA-positive ribbon-like lesions that replaced neuropil and extended along the hippocampal gray matter. We also identified dysplastic neurons infiltrating into the CA4 hilus of the hippocampus. Intense INA expression was present in the stroma as well as the cytoplasmic membrane of dysplastic neurons and their processes. While the invasiveness suggested a neoplasm, a relatively restrictive, either nodular or ribbon-like growth pattern with INA-positive abnormal neuropil suggested a hamartoma. Such quasi-tumors should be accommodated in the World Health Organization classification of tumors of the central nervous system, as are dysembryoplastic neuroepithelial tumor and Lhermitte-Duclos disease. PMID:26644357

  7. Intracorneal vacuoles in skin diseases with parakeratotic hyperkeratosis in the dog: a retrospective light-microscopy study of 111 cases (1973-2000).

    Science.gov (United States)

    Senter, David A; Scott, Danny W; Miller, William H; Erb, Hollis N

    2002-02-01

    Two recent case reports described a congenital keratinization defect (congenital follicular parakeratosis; CFP) in Rottweiler and Siberian Husky dogs. Skin biopsy specimens revealed marked parakeratosis targeting the hair follicle and numerous intracorneal vacuoles. A retrospective histopathological study was conducted on skin biopsy specimens from 111 dogs with diseases associated with parakeratotic hyperkeratosis to determine whether intracorneal vacuoles were present. Additional criteria evaluated were the size and location of the vacuoles and the degree of parakeratosis. Cases examined included dogs with primary idiopathic seborrhoea, necrolytic migratory erythema (NME), Malassezia dermatitis, zinc-responsive dermatosis, hereditary nasal hyperkeratosis of Labrador Retriever dogs, thallotoxicosis and CFP. Thirty-seven cases (37/111, 33%) had intracorneal vacuoles, including nine cases of primary idiopathic seborrhoea (9/29, 31%), 10 cases of NME (10/18, 56%), five cases of Malassezia dermatitis (5/19, 26%), five cases of zinc-responsive dermatosis (5/36, 14%), five cases of hereditary nasal hyperkeratosis (5/5, 100%) and three cases of CFP (3/3, 100%). If present, intracorneal vacuoles were found throughout all layers of the parakeratin. The sizes of intracorneal vacuoles varied among diseases, but large (> 5 microm) vacuoles only were present in CFP. Biopsies with a larger degree of parakeratosis were significantly more likely to have intracorneal vacuoles (P = dog, but large (> 5 microm) vacuoles are found only in CFP. PMID:11896970

  8. Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development.

    Science.gov (United States)

    Dolega, Monika E; Abeille, Fabien; Picollet-D'hahan, Nathalie; Gidrol, Xavier

    2015-06-01

    3D culture systems are a valuable tool for modeling morphogenesis and carcinogenesis of epithelial tissue in a structurally appropriate context. We present a novel approach for 3D cell culture based on a flow-focusing microfluidic system that encapsulates epithelial cells in Matrigel beads. As a model we use prostatic and breast cells and assay for development of acini, polarized cellular spheres enclosing lumen. Each individual bead on average acts as a single 3D cell culture compartment generating one acinus per bead. Compared to standard protocols microfluidics provides increased control over the environment leading to more a uniform acini population. The increased facility of bead manipulation allowed us to isolate single cells which are self-sufficient to fully develop into acini in presence of Matrigel. Furthermore, combination of our microfluidic approach with large particle FACS opens new avenues in high throughput screening on single acini or spheroids.

  9. Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after amino acid feeding

    DEFF Research Database (Denmark)

    Rojek, Aleksandra; Füchtbauer, Ernst-Martin; Füchtbauer, Annette C.;

    2013-01-01

    Aquaporin 11 (AQP11) is a protein channel expressed intracellularly in multiple organs, yet its physiological function is unclear. Aqp11 knockout (KO) mice die early due to malfunction of the kidney, a result of hydropic degeneration of proximal tubule cells. Here we report the generation of liver......-specific Aqp11 KO mice, allowing us to study the role of AQP11 protein in liver of mice with normal kidney function. The unchallenged liver-specific Aqp11 KO mice have normal longevity, their livers appeared normal, and the plasma biochemistries revealed only a minor defect in lipid handling. Fasting...... of the mice (24 h) induced modest dilatation of the rough endoplasmic reticulum (RER) in the periportal hepatocytes. Refeeding with standard mouse chow induced rapid generation of large RER-derived vacuoles in Aqp11 KO mice hepatocytes. Similar effects were observed following oral administration of pure...

  10. The Quest for Tissue Stem Cells in the Pancreas and Other Organs, and their Application in Beta-Cell Replacement

    OpenAIRE

    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...

  11. E2f1-deficient NOD/SCID mice have dry mouth due to a change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland.

    Science.gov (United States)

    Satoh, Keitaro; Narita, Takanori; Matsuki-Fukushima, Miwako; Okabayashi, Ken; Ito, Tatsuro; Senpuku, Hidenobu; Sugiya, Hiroshi

    2013-02-01

    Non-obese diabetic (NOD) mice have been used as a model for dry mouth. NOD mice lacking the gene encoding E2f1, a transcription factor, develop hyposalivation more rapidly progressively than control NOD mice. However, the model mice are associated with an underlying disease such as diabetes. We have now established E2f1-deficient NOD/severe combined immunodeficiency disease (NOD/SCID.E2f1(-/-)) mice to avoid the development of diabetes (Matsui-Inohara et al., Exp Biol Med (Maywood) 234(12):1525-1536, 2009). In this study, we investigated the pathophysiological features of dry mouth using NOD/SCID.E2f1(-/-) mice. In NOD/SCID.E2f1(-/-) mice, the volume of secreted saliva stimulated with pilocarpine is about one third that of control NOD/SCID mice. In behavioral analysis, NOD/SCID.E2f1(-/-) mice drank plenty of water when they ate dry food, and the frequency and time of water intake were almost double compared with control NOD/SCID mice. Histological analysis of submandibular glands with hematoxylin-eosin stain revealed that NOD/SCID.E2f1(-/-) mice have more ducts than NOD/SCID mice. In western blot analysis, the expression of aquaporin 5 (AQP5), a marker of acinar cells, in parotid and in submandibular glands of NOD/SCID.E2f1(-/-) mice was lower than in NOD/SCID mice. Immunohistochemical analysis of parotid and submandibular acini revealed that the localization of AQP5 in NOD/SCID.E2f1(-/-) mice differs from that in NOD/SCID mice; AQP5 was leaky and diffusively localized from the apical membrane to the cytosol in NOD/SCID.E2f1(-/-) mice. The ubiquitination of AQP5 was detected in submandibular glands of NOD/SCID.E2f1(-/-) mice. These findings suggest that the change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland cause the pathogenesis of hyposalivation in NOD/SCID.E2f1(-/-) mice.

  12. Transgenic tobacco plants expressing yeast-derived invertase in either the cytosol, vacuole or apoplast: a powerful tool for studying sucrose metabolism and sink/source interactions.

    Science.gov (United States)

    Sonnewald, U; Brauer, M; von Schaewen, A; Stitt, M; Willmitzer, L

    1991-07-01

    In higher plants sucrose plays a central roles with respect to both short-term storage and distribution of photoassimilates formed in the leaf. Sucrose is synthesized in the cytosol, transiently stored in the vacuole and exported via the apoplast. In order to elucidate the role of the different compartments with respect to sucrose metabolism, a yeast-derived invertase was directed into the cytosol and vacuole of transgenic tobacco plants. This was in addition to the targeting of yeast-derived invertase into the apoplast described previously. Vacuolar targeting was achieved by fusing an N-terminal portion (146 amino acids long) of the vacuolar protein patatin to the coding region of the mature invertase protein. Transgenic tobacco plants expressing the yeast-derived invertase in different subcellular compartments displayed dramatic phenotypic differences when compared to wild-type plants. All transgenic plants showed stunted growth accompanied by reduced root formation. Starch and soluble sugars accumulated in leaves indicating that the distribution of sucrose was impaired in all cases. Expression of cytosolic yeast invertase resulted in the accumulation of starch and soluble sugars in both very young (sink) and older (source) leaves. The leaves were curved, indicating a more rapid cell expansion or cell division at the upper side of the leaf. Light-green sectors with reduced photosynthetic activity were evenly distributed over the leaf surface. With the apoplastic and vacuolar invertase, the phenotypical changes induced only appear in older (source) leaves. The development of bleached and/or necrotic sectors was linked to the source state of a leaf. Bleaching followed the sink to source transition, starting at the rim of the leaf and moving to the base. The bleaching was paralleled by the inhibition of photosynthesis. PMID:1844880

  13. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    Science.gov (United States)

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis. PMID:27457979

  14. Protein kinase D regulates cell death pathways in experimental pancreatitis

    Directory of Open Access Journals (Sweden)

    Jingzhen eYuan

    2012-03-01

    Full Text Available Inflammation and acinar cell necrosis are two major pathological responses of acute pancreatitis, a serious disorder with no current therapies directed to its molecular pathogenesis. Serine/threonine protein kinase D family, which includes PKD/PKD1, PKD2, and PKD3, has been increasingly implicated in the regulation of multiple physiological and pathophysiological effects. We recently reported that PKD/PKD1, the predominant PKD isoform expressed in rat pancreatic acinar cells, mediates early events of pancreatitis including NF-kappaB activation and inappropriate intracellular digestive enzyme activation. In current studies, we investigated the role and mechanisms of PKD/PKD1 in the regulation of necrosis in pancreatic acinar cells by using two novel small molecule PKD inhibitors CID755673 and CRT0066101 and molecular approaches in in vitro and in vivo experimental models of acute pancreatitis. Our results demonstrated that both CID755673 and CRT0066101 are PKD-specific inhibitors and that PKD/PKD1 inhibition by either the chemical inhibitors or specific PKD/PKD1 siRNAs attenuated necrosis while promoting apoptosis induced by pathological doses of cholecystokinin-octapeptide (CCK in pancreatic acinar cells. Conversely, upregulation of PKD expression in pancreatic acinar cells increased necrosis and decreased apoptosis. We further showed that PKD/PKD1 regulated several key cell death signals including inhibitors of apoptotic proteins (IAPs, caspases, receptor-interacting protein kinase 1 (RIP1 to promote necrosis. PKD/PKD1 inhibition by CID755673 significantly ameliorated necrosis and severity of pancreatitis in an in vivo experimental model of acute pancreatitis. Thus, our studies indicate that PKD/PKD1 is a key mediator of necrosis in acute pancreatitis and that PKD/PKD1 may represent a potential therapeutic target in acute pancreatitis.

  15. Perlecan domain IV peptide stimulates salivary gland cell assembly in vitro.

    Science.gov (United States)

    Pradhan, Swati; Zhang, Chu; Jia, Xinqiao; Carson, Daniel D; Witt, Robert; Farach-Carson, Mary C

    2009-11-01

    Treatment of xerostomia would benefit from development of a functional implantable artificial salivary gland. Salivary gland tissue from surgical patients was assessed by histology and immunohistochemistry to establish the phenotype of normal salivary gland cells including the native basement membranes. Ductal and acinar cells were identified in tissue and cultured cells from dispersed tissue. High levels of laminin and perlecan/HSPG2 (heparan sulfate proteoglycan 2) were noted in basement membranes, and perlecan also was secreted and organized by cultured acinar populations, which formed lobular structures that mimicked intact glands when cultured on Matrigel or a bioactive peptide derived from domain IV of perlecan. On either matrix, large acini-like lobular structures grew and formed connections between the lobes. alpha-Amylase secretion was confirmed by staining and activity assay. Biomarkers, including tight junction protein E-cadherin and water channel protein aquaporin 5 found in tissue, were expressed in cultured acinar cells. Cells cultured on Matrigel or domain IV of perlecan peptide organized stress fibers and activated focal adhesion kinase. We report a novel technique to isolate acinar cells from human salivary gland and identify a human peptide sequence in perlecan that triggers differentiation of salivary gland cells into self-assembling acini-like structures that express essential biomarkers and which secrete alpha-amylase.

  16. Static Clathrin Assemblies at the Peripheral Vacuole-Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia.

    Science.gov (United States)

    Zumthor, Jon Paulin; Cernikova, Lenka; Rout, Samuel; Kaech, Andres; Faso, Carmen; Hehl, Adrian B

    2016-07-01

    Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of

  17. Static Clathrin Assemblies at the Peripheral Vacuole-Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia.

    Directory of Open Access Journals (Sweden)

    Jon Paulin Zumthor

    2016-07-01

    Full Text Available Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs, which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM. Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC, dynamin-related protein (GlDRP, and assembly polypeptide complex 2 (AP2 subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular

  18. New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy.

    Science.gov (United States)

    Regvar, Marjana; Eichert, Diane; Kaulich, Burkhard; Gianoncelli, Alessandra; Pongrac, Paula; Vogel-Mikus, Katarina; Kreft, Ivan

    2011-07-01

    Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.

  19. Distal myopathy with rimmed vacuoles: Report on clinical characteristics in 23 cases

    Directory of Open Access Journals (Sweden)

    Nalini A

    2010-01-01

    Full Text Available Background: Distal myopathy with rimmed vacuoles (DMRV is an autosomal recessive (AR myopathy characterized clinically by the preferential involvement of the tibialis anterior and has been reported predominantly in the Japanese population. Materials and Methods: A case series of DMRV patients seen over a period of 3 years at a tertiary national referral center for neurological disorders in south India. Results: We describe the clinical characteristics, muscle magnetic resonance imaging (MRI findings and classical histopathological feature in 23 patients. There were 12 men and 11 women. Mean age of onset was 27.04 ± 6.35 years (10-39 years. Onset was in the second or third decade in a majority. Mean age at presentation was 33.95 ± 6.35 years (25-48 years. Mean duration of illness was 6.74 ± 4.8 years (1-18 years. Consanguinity was reported in eight (34.8% patients. The predominant and initial manifestation was bilateral foot drop in all patients. Muscle MRI demonstrated classical involvement of the anterior compartment muscles of the lower legs and the posterior compartment muscles of the thighs and the quadriceps was normal in all. Muscle histopathology showed numerous fibers containing rimmed vacuoles. Necrotic fibers or phagocytosis or regenerating fibers were rarely noted or were absent. Conclusions: DMRV is a rare AR myopathy. The disorder presents as progressive foot drop and hence has many differential diagnoses. It is easily mistaken as neuropathy of hereditary nature and hence it is extremely important to recognize the preferential muscle involvement and characterize the phenotype. This is the first report from India with patients having characteristic phenotype of Nonaka′s/AR hereditary inclusion body myopathy with quadriceps sparing, and all were confirmed by histopathology.

  20. Ascl3 marks adult progenitor cells of the mouse salivary gland.

    Science.gov (United States)

    Rugel-Stahl, Anastasia; Elliott, Marilyn E; Ovitt, Catherine E

    2012-05-01

    The Ascl3 transcription factor marks a subset of salivary gland duct cells present in the three major salivary glands of the mouse. In vivo, these cells generate both duct and secretory acinar cell descendants. Here, we have analyzed whether Ascl3-expressing cells retain this multipotent lineage potential in adult glands. Cells isolated from mouse salivary glands were cultured in vitro as non-adherent spheres. Lineage tracing of the Ascl3-expressing cells within the spheres demonstrates that Ascl3+ cells isolated from adult glands remain multipotent, generating both duct and acinar cell types in vitro. Furthermore, we demonstrate that the progenitor cells characterized by Keratin 5 expression are an independent population from Ascl3+ progenitor cells. We conclude that the Ascl3+ cells are intermediate lineage-restricted progenitor cells of the adult salivary glands.

  1. LOXL2 induces aberrant acinar morphogenesis via ErbB2 signaling

    NARCIS (Netherlands)

    J. Chang (Jufang); M.M. Nicolau (Monica); T.R. Cox (Thomas); D. Wetterskog (Daniel); J.W.M. Martens (John); H. E Barker (Holly); J.T. Erler (Janine)

    2013-01-01

    textabstractIntroduction: 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 expr

  2. The I-BAR protein Ivy1 is an effector of the Rab7 GTPase Ypt7 involved in vacuole membrane homeostasis

    NARCIS (Netherlands)

    Numrich, Johannes; Péli-Gulli, Marie-Pierre; Arlt, Henning; Sardu, Alessandro; Griffith, Janice; Levine, Tim; Engelbrecht-Vandré, Siegfried; Reggiori, Fulvio; De Virgilio, Claudio; Ungermann, Christian

    2015-01-01

    Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show

  3. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

    NARCIS (Netherlands)

    Korstanje, Ron; Desai, Jigar; Lazar, Gloria; King, Benjamin; Rollins, Jarod; Spurr, Melissa; Joseph, Jamie; Kadambi, Sindhuja; Li, Yang; Cherry, Allison; Matteson, Paul G.; Paigen, Beverly; Millonig, James H.

    2008-01-01

    Korstanje R, Desai J, Lazar G, King B, Rollins J, Spurr M, Joseph J, Kadambi S, Li Y, Cherry A, Matteson PG, Paigen B, Millonig JH. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects. Physiol Genomics 35: 296-30

  4. The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development.

    Science.gov (United States)

    Van Son, Le; Tiedemann, Jens; Rutten, Twan; Hillmer, Stefan; Hinz, Giselbert; Zank, Thorsten; Manteuffel, Renate; Bäumlein, Helmut

    2009-11-01

    BURP domain proteins comprise a broadly distributed, plant-specific family of functionally poorly understood proteins. VfUSP (Vicia faba Unknown Seed Protein) is the founding member of this family. The BURP proteins are characterized by a highly conserved C-terminal protein domain with a characteristic cysteine-histidine pattern. The Arabidopsis genome contains five BURP-domain encoding genes. Three of them are similar to the non-catalytic beta-subunit of the polygalacturonase of tomato and form a distinct subgroup. The remaining two genes are AtRD22 and AtUSPL1. The deduced product of AtUSPL1 is similar in size and sequence to VfUSP and that of the Brassica napus BNM2 gene which is expressed during microspore-derived embryogenesis. The protein products of BURP genes have not been found, especially that of VfUSP despite a great deal of interest arising from copious transcription of the gene in seeds. Here, we demonstrate that VfUSP and AtUSPL1 occur in cellular compartments essential for seed protein synthesis and storage, like the Golgi cisternae, dense vesicles, prevaculoar vesicles and the protein storage vacuoles in the parenchyma cells of cotyledons. Ectopic expression of AtUSPL1 leads to a shrunken seed phenotype; these seeds show structural alterations in their protein storage vacuoles and lipid vesicles. Furthermore, there is a reduction in the storage protein content and a perturbation in the seed fatty acid composition. However, loss of AtUSP1 gene function due to T-DNA insertions does not lead to a phenotypic change under laboratory conditions even though the seeds have less storage proteins. Thus, USP is pertinent to seed development but its role is likely shared by other proteins that function well enough under the laboratory growth conditions. PMID:19639386

  5. Recent advances in stem cell research for the treatment of diabetes

    OpenAIRE

    Noguchi, Hirofumi

    2009-01-01

    The success achieved over the last decade with islet transplantation has intensified interest in treating diabetes, not only by cell transplantation, but also by stem cells. The formation of insulin-producing cells from pancreatic duct, acinar, and liver cells is an active area of investigation. Protocols for the in vitro differentiation of embryonic stem (ES) cells based on normal developmental processes, have generated insulin-producing cells, though at low efficiency and without full respo...

  6. Estrategias para la diferenciación in vitro de células ES de ratón a células acinares pancreáticas

    OpenAIRE

    Rovira Clusellas, Meritxell

    2007-01-01

    Las patologías más importantes del páncreas exocrino, como la pancreatitis crónica (PC) o el cáncer de páncreas, representan un gran problema de salud pública en Europa. En la PC, el tejido acinar es substituido por complejos ductales. Además, es difícil mantener el fenotipo diferenciado de las células acinares en cultivo ya que sufren una transdiferenciación acinar-ductal.Las células madre embrionarias (ES) de ratón han sido utilizadas en la última década para generar in vitro células comple...

  7. Rimmed vacuoles with beta-amyloid and ubiquitinated filamentous deposits in the muscles of patients with long-standing denervation (postpoliomyelitis muscular atrophy): similarities with inclusion body myositis.

    Science.gov (United States)

    Semino-Mora, C; Dalakas, M C

    1998-10-01

    In the chronically denervated muscles of patients with prior paralytic poliomyelitis, there are secondary myopathic features, including endomysial inflammation and rare vacuolated fibers. To assess the frequency and characteristics of the vacuoles and their similarities with those seen in inclusion body myositis (IBM), we examined 58 muscle biopsy specimens from patients with prior paralytic poliomyelitis for (1) the presence of rimmed vacuoles; (2) acid-phosphatase reactivity; (3) Congo-red-positive amyloid deposits; (4) electron microscopy, searching for tubulofilaments; and (5) immunoelectron microscopy, using antibodies against beta-amyloid and ubiquitin. We found vacuolated muscle fibers in 18 of 58 (31%) biopsies, with a mean frequency of 2.06 +/- 0.42 fibers per specimen. The vacuoles contained acid phosphatase-positive material in 6 of the 18 (33.30%) specimens and stained positive for Congo red in five (27.80%). By immunoelectron microscopy, the vacuoles contained 5.17 +/- 0.13 nm fibrils and 14.9 +/- 0.31 nm filaments that immunoreacted with antibodies to beta-amyloid and ubiquitin in a pattern identical to the one seen in IBM. We conclude that vacuolated muscle fibers containing filamentous inclusions positive for amyloid and ubiquitin are not unique to IBM and the other vacuolar myopathies but can also occur in a chronic neurogenic condition, such as postpoliomyelitis. The chronicity of the underlying disease, rather than the cause, may lead to vacuolar formation, amyloid deposition, and accumulation of ubiquitinated filaments.

  8. Expression of 87 kD protein in the broth culture filtrate of Helicobacter pylori and its association with the vacuolating effect

    Institute of Scientific and Technical Information of China (English)

    SHI Li; YIE Gui-an; NAN Qing-zhen; SUN Yong; ZHANG Ya-li; ZHANG Zhen-shu; ZHOU Dian-yuan

    2001-01-01

    To study the vacuolating effect of Helicobacter pylori(H.pylori). Method: The vacuolating effect and its relationship with vacuolating cytotoxin antigen (an 87 kD protein) were investigated by the method of cytotoxic test, SDS-PAGE and scanning. Result: Of the 62 clinical isolates, 43 strains were H.pylori (Toxin+) with vacuolating effect, while the others were H.pylori (Toxin-) without vacuolating effect. Altogether 78.26%(36/46) patients with peptic ulcer were infected with H.pylori (Toxin+) strains, and only 42.86%(6/14) who had gastritis were infected with H.pylori (Toxin+) strains, with significant difference between them(χ2=4.83,P<0.05). A protein with relativemolecular mass of 87 kD was identified in the broth culture filter(BCF) of 30.23% H. Pylori (Toxin+) strains (13/43) but in none of that of H.pylori (Toxin-) strains, and the difference was statistically significant(P<0.05). There was a significant and concordant relationship between the OD value of the protein band and the titer of vacuolating activity of H.pylori (Toxin+) (r=0.67 and P<0.05 by linear regression analysis). Conclusion: H.pylori (Toxin+) were more often associated with peptic ulcerous diseases than with gastritis diseases. The vacuolating effect of H.pylori (Toxin+) may be caused by the 87 kD protein.

  9. Role of vacuolar membrane proton pumps in the acidification of protein storage vacuoles following germination.

    Science.gov (United States)

    Wilson, Karl A; Chavda, Burzin J; Pierre-Louis, Gandhy; Quinn, Adam; Tan-Wilson, Anna

    2016-07-01

    During soybean (Glycine max (L.) Merrill) seed development, protease C1, the proteolytic enzyme that initiates breakdown of the storage globulins β-conglycinin and glycinin at acidic pH, is present in the protein storage vacuoles (PSVs), the same subcellular compartments in seed cotyledons where its protein substrates accumulate. Actual proteolysis begins to be evident 24 h after seed imbibition, when the PSVs become acidic, as indicated by acridine orange accumulation visualized by confocal microscopy. Imidodiphosphate (IDP), a non-hydrolyzable substrate analog of proton-translocating pyrophosphatases, strongly inhibited acidification of the PSVs in the cotyledons. Consistent with this finding, IDP treatment inhibited mobilization of β-conglycinin and glycinin, the inhibition being greater at 3 days compared to 6 days after seed imbibition. The embryonic axis does not appear to play a role in the initial PSV acidification in the cotyledon, as axis detachment did not prevent acridine orange accumulation three days after imbibition. SDS-PAGE and immunoblot analyses of cotyledon protein extracts were consistent with limited digestion of the 7S and 11S globulins by protease C1 starting at the same time and proceeding at the same rate in detached cotyledons compared to cotyledons of intact seedlings. Embryonic axis removal did slow down further breakdown of the storage globulins by reactions known to be catalyzed by protease C2, a cysteine protease that normally appears later in seedling growth to continue the storage protein breakdown initiated by protease C1. PMID:27043965

  10. The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

    DEFF Research Database (Denmark)

    Müller, Oliver; Bayer, Martin J; Peters, Christopher;

    2002-01-01

    The fusion of cellular membranes comprises several steps; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast...... vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events....... The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step...

  11. Taxol-induced paraptosis-like A549 cell death is not senescence

    Science.gov (United States)

    Wang, Chao-yang; Chen, Tong-Sheng

    2011-03-01

    Our previous studies have shown that taxol, a potent anticancer agent, induces caspase-independent cell death and cytoplasmic vacuolization in human lung cancer cells. However, the mechanisms of taxol-induced cytoplasmic vacuolization are poorly understood. Cytoplasmic vacuolization have been reported to be a characteristic of cell senescence. Here, we employed confocal fluorescence microscopy imaging to study the reversibility of taxol-induced cytoplasmic vacuolization and whether taxol triggers senescence in A549 cells. We found that taxol-induced cytoplasmic vacuolization at 6 or 9 h after treatment with taxol did not decrease but increase at 24 h or 72 h after refreshing the culture medium without taxol, indicating taxol-induced cytoplasmic vacuolization is irreversible. We used SA-β-Gal (senescence-associated β-galactosidase) to assess whether taxol-induced cell death in cytoplasmic vacuolization fashion is senescence, and found that hydrogen peroxide (H2O2)-treated, but not taxol-treated cells is significantly stained by the SA-β-Gal, a senescence testing kit, indicating that the form of taxol-induced cell death is not senescence.

  12. Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

    Science.gov (United States)

    Balsamo, R A; Uribe, E G

    1988-02-01

    Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces. PMID:24226399

  13. Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

    Science.gov (United States)

    Pulcini, Serena; Staines, Henry M; Lee, Andrew H; Shafik, Sarah H; Bouyer, Guillaume; Moore, Catherine M; Daley, Daniel A; Hoke, Matthew J; Altenhofen, Lindsey M; Painter, Heather J; Mu, Jianbing; Ferguson, David J P; Llinás, Manuel; Martin, Rowena E; Fidock, David A; Cooper, Roland A; Krishna, Sanjeev

    2015-01-01

    Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

  14. Chronic ingestion of 2-deoxy-D-glucose induces cardiac vacuolization and increases mortality in rats

    International Nuclear Information System (INIS)

    Calorie restriction (CR), the purposeful reduction of energy intake with maintenance of adequate micronutrient intake, is well known to extend the lifespan of laboratory animals. Compounds like 2-deoxy-D-glucose (2DG) that can recapitulate the metabolic effects of CR are of great interest for their potential to extend lifespan. 2DG treatment has been shown to have potential therapeutic benefits for treating cancer and seizures. 2DG has also recapitulated some hallmarks of the CR phenotype including reduced body temperature and circulating insulin in short-term rodent trials, but one chronic feeding study in rats found toxic effects. The present studies were performed to further explore the long-term effects of 2DG in vivo. First we demonstrate that 2DG increases mortality of male Fischer-344 rats. Increased incidence of pheochromocytoma in the adrenal medulla was also noted in the 2DG treated rats. We reconfirm the cardiotoxicity of 2DG in a 6-week follow-up study evaluating male Brown Norway rats and a natural form of 2DG in addition to again examining effects in Fischer-344 rats and the original synthetic 2DG. High levels of both 2DG sources reduced weight gain secondary to reduced food intake in both strains. Histopathological analysis of the hearts revealed increasing vacuolarization of cardiac myocytes with dose, and tissue staining revealed the vacuoles were free of both glycogen and lipid. We did, however, observe higher expression of both cathepsin D and LC3 in the hearts of 2DG-treated rats which indicates an increase in autophagic flux. Although a remarkable CR-like phenotype can be reproduced with 2DG treatment, the ultimate toxicity of 2DG seriously challenges 2DG as a potential CR mimetic in mammals and also raises concerns about other therapeutic applications of the compound.

  15. Selective functionalization of nanofiber scaffolds to regulate salivary gland epithelial cell proliferation and polarity.

    Science.gov (United States)

    Cantara, Shraddha I; Soscia, David A; Sequeira, Sharon J; Jean-Gilles, Riffard P; Castracane, James; Larsen, Melinda

    2012-11-01

    Epithelial cell types typically lose apicobasal polarity when cultured on 2D substrates, but apicobasal polarity is required for directional secretion by secretory cells, such as salivary gland acinar cells. We cultured salivary gland epithelial cells on poly(lactic-co-glycolic acid) (PLGA) nanofiber scaffolds that mimic the basement membrane, a specialized extracellular matrix, and examined cell proliferation and apicobasal polarization. Although cells proliferated on nanofibers, chitosan-coated nanofiber scaffolds stimulated proliferation of salivary gland epithelial cells. Although apicobasal cell polarity was promoted by the nanofiber scaffolds relative to flat surfaces, as determined by the apical localization of ZO-1, it was antagonized by the presence of chitosan. Neither salivary gland acinar nor ductal cells fully polarized on the nanofiber scaffolds, as determined by the homogenous membrane distribution of the mature tight junction marker, occludin. However, nanofiber scaffolds chemically functionalized with the basement membrane protein, laminin-111, promoted more mature tight junctions, as determined by apical localization of occludin, but did not affect cell proliferation. To emulate the multifunctional capabilities of the basement membrane, bifunctional PLGA nanofibers were generated. Both acinar and ductal cell lines responded to signals provided by bifunctional scaffolds coupled to chitosan and laminin-111, demonstrating the applicability of such scaffolds for epithelial cell types.

  16. Increased heat shock protein 70 expression in the pancreas of rats with endotoxic shock

    Institute of Scientific and Technical Information of China (English)

    Xue-Lian Wang; Ying Li; Jin-Song Kuang; Yue Zhao; Pei Liu

    2006-01-01

    AIM: To investigate the ultra-structural changes and heat shock protein 70 (HSP70) expression in the pancreas of rats with endotoxic shock and to detect their possible relationship.METHODS: A total of 33 Wistar rats were randomly divided into three groups: control group (given normal saline), small dose lipopolysaccharide (LPS) group (given LPS 5 mg/kg) and large dose LPS group (given LPS 10mg/kg). Pancreas was explanted to detect the ultrastructural changes by TEM and the HSP70 expression by immunohistochemistry and Western blot.RESULTS: Rats given small doses of LPS showed swelling and loss of mitochondrial cristae of acinar cells and increased number of autophagic vacuoles in the cytoplasm of acinar cells. Rats given large doses of LPS showed swelling, vacuolization, and obvious myeloid changes of mitochondrial cristae of acinar cells, increased number of autophagic vacuoles in the cytoplasm of acinar cells. HSP70 expression was increased compared to the control group (P<0.05).CONCLUSION: Small doses of LPS may induce stronger expression of HSP70, promote autophagocytosis and ameliorate ultra-structural injuries.

  17. Computed tomographical findings of skeletal muscles in rimmed vacuole type distal myopathy

    Energy Technology Data Exchange (ETDEWEB)

    Kunimoto, Masanari; Kawai, Mitsuru; Goto, Jun; Nakano, Imaharu

    1987-03-01

    Skeletal muscle CT scans of three patients with biopsy-proven rimmed vacuole type distal myopathy(RVDM) from two unrelated families showed unique involvement pattern of the lower extremities. Parents of the patients in both families are first cousins. T.Y. is a 36-year-old woman who noticed mild difficulty in walking at 34 years of age. Now she shows waddling but still indpendent gait. Manual muscle test (MMT) revealed the following results: fair (3+/5) for hip flexion, 3 for hip extension, 3 for knee flexion, normal (5/5) for knee extension, poor (2/5) for ankle dorsi-flexion, good (4/5) for ankle plantar flexion. T.M., a 34-year-old younger sister of T.Y., started dragging her feet on gait at age 27. She could walk neither on toes nor heels. At present, she can walk only with support. MMT showed the following: 3- and trace (1/5) for hip flexion and extension, 3- and 4 for knee flexion and extension, 1 for ankle plantar- and dorsiflexion. K.W., a 33-year-old woman, began to drag her foot tips in walk and became unable to walk on toes at age 21. The leg weakness progressed into the wheelchair-ridden state at age 30. MMT gave the following results: 1 for hip flexion and extension, 2 and 4 for knee flexion and extension, zero for plantar- and dorsi-flexion. The most impressive CT findings common to these three patients are prominent contrast between the quadriceps muscles and the adductor- and hamstring-group: the former is markedly well preserved even in the most advanced patient (K.W.) while the latter are diffusely and severely affected even in the least affected patient (T.Y.). This finding well coincides with the results of MMT: the knee extensor (quadriceps muscles) fairly well keeps its strength even in the most advanced patient but the knee flexors (adductors and hamstrings) are definitely affected in the early stage of the condition. (J.P.N.).

  18. Effect of Trimethyltin Chloride on Slow Vacuolar (SV Channels in Vacuoles from Red Beet (Beta vulgaris L. Taproots.

    Directory of Open Access Journals (Sweden)

    Zenon Trela

    Full Text Available In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl on the slow vacuolar (SV channels in vacuoles from red beet (Beta vulgaris L. taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10% decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5 Met3SnCl is a non-dissociated (more lipophilic compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.

  19. Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots.

    Science.gov (United States)

    Trela, Zenon; Burdach, Zbigniew; Siemieniuk, Agnieszka; Przestalski, Stanisław; Karcz, Waldemar

    2015-01-01

    In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met3SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel. PMID:26317868

  20. Latent and persistent lethal injury in mouse salivary gland cells following gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Sasaki, T.

    1976-07-01

    Newly synthesized DNA in previously irradiated and isoproterenol-stimulated mouse salivary gland cells was found to be quickly degraded when the stimulation for DNA synthesis was given 10 days after a dose of 1000 rad ..gamma.. radiation. The degradation of the DNA was due to degeneration of acinar cells prior to mitosis. When the stimulation with isoproterenol was given 1 or 3 months after irradiation, DNA degradation in parotids was not detectable. An autoradiographic analysis revealed, however, that about half of the acinar cells labeled with tritiated thymidine were eliminated from irradiated parotids in a few days, even when the stimulation with isoproterenol was given 3 months after irradiation. This indicates that irradiation of mouse salivary gland cells produced latent lethal damage and that this damage is unmasked by the stimulation for DNA synthesis and cell division.

  1. Analyzing Ca(2+) dynamics in intact epithelial cells using spatially limited flash photolysis.

    Science.gov (United States)

    Almassy, Janos; Yule, David I

    2013-01-01

    The production of saliva by parotid acinar cells is stimulated by Ca(2+) activation of Cl(-) and K(+) channels located in the apical plasma membrane of these polarized cells. Here we describe a paradigm for the focal photorelease of either Ca(2+) or an inositol 1,4,5 trisphosphate (InsP(3)) analog. The protocol is designed to be useful for investigating subcellular Ca(2+) dynamics in polarized cells with minimal experimental intervention. Parotid acinar cells are loaded with cell-permeable versions of the caged precursors (NP-EGTA-AM or Ci-InsP(3)/PM). Photolysis is accomplished using a spatially limited, focused diode laser, but the experiment can be readily modified to whole-field photolysis using a xenon flash lamp.

  2. Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

    Directory of Open Access Journals (Sweden)

    Alberto Muñoz

    Full Text Available The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW and PAF96 (RKKAAA, were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i its interaction with the cell envelope; (ii its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the

  3. Rap1 integrates tissue polarity, lumen formation, and tumorigenicpotential in human breast epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Itoh, Masahiko; Nelson, Celeste M.; Myers, Connie A.; Bissell,Mina J.

    2006-09-29

    Maintenance of apico-basal polarity in normal breast epithelial acini requires a balance between cell proliferation, cell death, and proper cell-cell and cell-extracellular matrix signaling. Aberrations in any of these processes can disrupt tissue architecture and initiate tumor formation. Here we show that the small GTPase Rap1 is a crucial element in organizing acinar structure and inducing lumen formation. Rap1 activity in malignant HMT-3522 T4-2 cells is appreciably higher than in S1 cells, their non-malignant counterparts. Expression of dominant-negative Rap1 resulted in phenotypic reversion of T4-2 cells, led to formation of acinar structures with correct apico-basal polarity, and dramatically reduced tumor incidence despite the persistence of genomic abnormalities. The resulting acini contained prominent central lumina not observed when other reverting agents were used. Conversely, expression of dominant-active Rap1 in T4-2 cells inhibited phenotypic reversion and led to increased invasiveness and tumorigenicity. Thus, Rap1 acts as a central regulator of breast architecture, with normal levels of activation instructing apical polarity during acinar morphogenesis, and increased activation inducing tumor formation and progression to malignancy.

  4. Virulence-related Mycobacterium avium subsp hominissuis MAV_2928 gene is associated with vacuole remodeling in macrophages

    Directory of Open Access Journals (Sweden)

    Vogt Steven

    2010-04-01

    Full Text Available Abstract Background Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928 homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion. Results MAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles with M. hominissuis wild-type in the concentration of zinc, manganese, calcium and potassium. Conclusion The results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.

  5. Hepatocyte vacuolation and autolytic changes in the liver of pilot whales, Globicephala melas, stranded on Cape Cod, MA, USA.

    Science.gov (United States)

    Moore, M J; Stegeman, J J

    1996-07-16

    Most cetacea available for internal sampling in recent times have died through mass or single stranding events. It is important to know how the time elapsed between death and sampling affect quality of tissues. This study evaluated histological quality in the liver of long-finned pilot whales that either died or were euthanased after mass stranding events. Histological detection of significant autolysis was found in animals when 2 or more hours elapsed between death and sampling. In addition, hepatocytes often had marked idiopathic cytoplasmic vacuolation that did not stain with hematoxylin and eosin. The extent of this vacuolation did not show any correlation with time between death and sampling, but did appear more often in animals of greater total length. These observations suggest that when animals die or are euthanased at a single or mass stranding, every effort should be made to obtain samples as soon as possible, although meaningful histological observations can still be made in the presence of significant autolysis. These data also suggest that a multi-disciplinary study should be conducted to determine whether increasing autolysis is associated with changes in the organic chemical residues, molecular biology, histopathology and microbiology of those tissues. PMID:8685702

  6. Anionic Sites, Fucose Residues and Class I Human Leukocyte Antigen Fate During Interaction of Toxoplasma gondii with Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Stumbo Ana Carolina

    2002-01-01

    Full Text Available Toxoplasma gondii invades and proliferates in human umbilical vein endothelial cells where it resides in a parasitophorous vacuole. In order to analyze which components of the endothelial cell plasma membrane are internalized and become part of the parasitophorous vacuole membrane, the culture of endothelial cells was labeled with cationized ferritin or UEA I lectin or anti Class I human leukocytte antigen (HLA before or after infection with T. gondii. The results showed no cationized ferritin and UEA I lectin in any parasitophorous vacuole membrane, however, the Class I HLA molecule labeling was observed in some endocytic vacuoles containing parasite until 1 h of interaction with T. gondii. After 24 h parasite-host cell interaction, the labeling was absent on the vacuolar membrane, but presents only in small vesicles near parasitophorous vacuole. These results suggest the anionic site and fucose residues are excluded at the time of parasitophorous vacuole formation while Class I HLA molecules are present only on a minority of Toxoplasma-containig vacuoles.

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

    Directory of Open Access Journals (Sweden)

    Anja Marciniak

    Full Text Available 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.

  8. Myoepithelial cells in pathology

    Directory of Open Access Journals (Sweden)

    N Balachander

    2015-01-01

    Full Text Available Myoepithelial cells are a normal constituent of the salivary acini and ducts and are found between the epithelial cells and the basement membrane. Microscopically myoepithelial cells are thin and spindle-shaped and ultrastructurally they possess a number of Cytoplasmic processes that extend between and over the acinar and ductal-lining cells, and they show features of both smooth muscle and epithelium. They play a vital role during expulsion of saliva and regulates the electrolytic exchange. They also perform as tumor suppressors and are considered to play a very important role in differentiation of various salivary gland tumors and help in the diagnosis of tumors. Neoplastic myoepithelial cells in both benign and malignant tumors can take numerous forms including epithelioid, plasmacytoid, spindle and clear cell variant, and this variability largely accounts for difficulties in histopathological diagnosis.

  9. Vacúolos de gás e flutuação em Difflugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea Gas vacuoles and flotation in Diffugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea

    Directory of Open Access Journals (Sweden)

    Vladimir Stolzenberg Torres

    1996-01-01

    Full Text Available The natural formation of gas vacuoles as a method of locomotion is described for Difflugia mitriformis Wallich, 1984. These vacuoles may contain different compositions of gases, basicly carbodioxyde or oxigen, with a membranous limitation similar or identical to other types of vacuoles. Those vacuoles are utilised by the organism as a mode of dislocation frorn the bottom to the water surface by flotation permiting better conditions for the survival of the individual, with the consequence of the perpetuance of the taxon.

  10. Legionella pneumophila infection of Drosophila S2 cells induces only minor changes in mitochondrial dynamics.

    Directory of Open Access Journals (Sweden)

    Elizabeth Wen Sun

    Full Text Available During infection of cells by Legionella pneumophila, the bacterium secretes a large number of effector proteins into the host cell cytoplasm, allowing it to alter many cellular processes and make the vacuole and the host cell into more hospitable environments for bacterial replication. One major change induced by infection is the recruitment of ER-derived vesicles to the surface of the vacuole, where they fuse with the vacuole membrane and prevent it from becoming an acidified, degradative compartment. However, the recruitment of mitochondria to the region of the vacuole has also been suggested by ultrastructural studies. In order to test this idea in a controlled and quantitative experimental system, and to lay the groundwork for a genome-wide screen for factors involved in mitochondrial recruitment, we examined the behavior of mitochondria during the early stages of Legionella pneumophila infection of Drosophila S2 cells. We found that the density of mitochondria near vacuoles formed by infection with wild type Legionella was not different from that found in dotA(- mutant-infected cells during the first 4 hours after infection. We then examined 4 parameters of mitochondrial motility in infected cells: velocity of movement, duty cycle of movement, directional persistence and net direction. In the 4 hours following infection, most of these measures were indistinguishable between wild type and dotA(-.infection. However, wild type Legionella did induce a modest shift in the velocity distribution toward faster movement compared dotA(- infection, and a small downward shift in the duty cycle distribution. In addition, wild type infection produced mitochondrial movement that was biased in the direction of the bacterial vacuole relative to dotA-, although not enough to cause a significant accumulation within 10 um of the vacuole. We conclude that in this host cell, mitochondria are not strongly recruited to the vacuole, nor is their motility

  11. Identification of the Helicobacter pylori VacA Toxin Domain Active in the Cell Cytosol

    Science.gov (United States)

    de Bernard, Marina; Burroni, Daniela; Papini, Emanuele; Rappuoli, Rino; Telford, John; Montecucco, Cesare

    1998-01-01

    Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles which originate from massive swelling of membranous compartments at late stages of the endocytic pathway. When expressed in the cytosol, VacA induces vacuolization as it does when added from outside. This and other evidence indicate that VacA is a toxin capable of entering the cell cytosol, where it displays its activity. In this study, we have used cytosolic expression to identify the portion of the toxin molecule responsible for the vacuolating activity. VacA mutants with deletions at the C and N termini were generated, and their activity was analyzed upon expression in HeLa cells. We found that the vacuolating activity of VacA resides in the amino-terminal region, the whole of which is required for its intracellular activity. PMID:9826387

  12. Haemophilus influenzae triggers autophagy in HEp-2 cells.

    Science.gov (United States)

    Espinoza-Mellado, María del Rosario; Reyes-Picaso, Carolina; Garcés-Pérez, Miriam S; Jardón-Serrano, Cynthia V; López-Villegas, Edgar O; Giono-Cerezo, Silvia

    2016-03-01

    The MAP-LC3 system regulates the intracellular formation of autophagy-associated vacuoles. These vacuoles contain the LC3 protein; thus it has been utilized as a marker to identify autophagosomes. The aim of our study was to investigate whether Haemophilus influenzae strains and their supernatants could activate autophagy in human larynx carcinoma cell line (HEp-2). We demonstrate that higher expression of the LC3B-II protein was induced, particularly by nontypeable Haemophilus influenzae (NTHi) 49766 and by supernatants, containing <50 kDa proteins, of both strains. Ultrastructural studies demonstrate vacuoles with a double membrane and/or membrane material inside, showing similar features to those of autophagic vacuoles. Together, our findings demonstrate that H. influenzae strains and their supernatants trigger an autophagic process.

  13. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    Science.gov (United States)

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  14. Cerulein Pancreatitis: Oxidative Stress, Inflammation, and Apoptosis

    OpenAIRE

    Kim, Hyeyoung

    2008-01-01

    Cerulein pancreatitis is similar to human edematous pancreatitis, manifesting with dysregulation of digestive enzyme production and cytoplasmic vacuolization, the death of acinar cells, edema formation, and infiltration of inflammatory cells into the pancreas. Reactive oxygen species are involved in nuclear factor-κB activation, cytokine expression, apoptosis and pathogenesis of pancreatitis. There is recent evidence that cerulein activates NADPH oxidase, which is a major source of reactive o...

  15. Primary observations of the existence of Fas-like cytoplasmic death factor in plant cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The main activity of Fas is to trigger cytoplasm death program in animal cells. In G2 pea, vacuole plays a pivotal role in inducing cell death in the cytoplasm of longday (LD) grown apical meristem cells. Expression patterns of the Fas in G2 pea cells revealed that the Fas is mainly localized in the vacuole of cells undergoing programmed cell death (PCD). The Fas expression is corresponding to the initiation of menadione-induced PCD in tobacco protoplasts.The results suggest the existence of the Fas-like mediated cytoplasmic death pathway in plant cells.``

  16. A multifunctional 3D co-culture system for studies of mammary tissue morphogenesis and stem cell biology.

    Directory of Open Access Journals (Sweden)

    Jonathan J Campbell

    Full Text Available Studies on the stem cell niche and the efficacy of cancer therapeutics require complex multicellular structures and interactions between different cell types and extracellular matrix (ECM in three dimensional (3D space. We have engineered a 3D in vitro model of mammary gland that encompasses a defined, porous collagen/hyaluronic acid (HA scaffold forming a physiologically relevant foundation for epithelial and adipocyte co-culture. Polarized ductal and acinar structures form within this scaffold recapitulating normal tissue morphology in the absence of reconstituted basement membrane (rBM hydrogel. Furthermore, organoid developmental outcome can be controlled by the ratio of collagen to HA, with a higher HA concentration favouring acinar morphological development. Importantly, this culture system recapitulates the stem cell niche as primary mammary stem cells form complex organoids, emphasising the utility of this approach for developmental and tumorigenic studies using genetically altered animals or human biopsy material, and for screening cancer therapeutics for personalised medicine.

  17. P2Y2 nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells.

    Science.gov (United States)

    El-Sayed, Farid G; Camden, Jean M; Woods, Lucas T; Khalafalla, Mahmoud G; Petris, Michael J; Erb, Laurie; Weisman, Gary A

    2014-07-01

    Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5β1 integrin and the Rho GTPase Cdc42.

  18. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion

    DEFF Research Database (Denmark)

    Ungermann, C; von Mollard, G F; Jensen, Ole Nørregaard;

    1999-01-01

    Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion...

  19. Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole

    NARCIS (Netherlands)

    Simeon, Angela; Klei, Ida J. van der; Veenhuis, Marten; Wolf, Dieter H.

    1992-01-01

    Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumu

  20. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    Directory of Open Access Journals (Sweden)

    Gal Winter

    Full Text Available 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.

  1. Studies on the development of male cells of third generation of the stratosphere radiative millet

    International Nuclear Information System (INIS)

    The developmental process of male cells of millet (Setaria italica), the third generation of the stratosphere radiative treatment SP3 and CK3, was studied. The results show that the normal process begins with archesporial cell and undergoes stages of primary and secondary sporogenous cell, microspore mother cell, dyad, tetrad, central nucleus microspore, vacuolated microspore, mature microspore, two-cell pollen and three-cell mature pollen. Among them, the formation of tetrad belongs to successive type. The situation of abnormal development of male cells is as follows: microspore mother cell can't enter into meiosis because of intense vacuolation, shrink and disintegration of its cytoplasm; although vacuolated microspore mother cell can enter into meiosis, it can't form normal dyad and degenerate in the middle process; dyad and tetrad become vacuolated and can't develop normally; cytoplasm of microspore shrinks around the nucleus at the stage of central nucleus microspore, the shape of microspore is twisted into crescent or irregular shape, at last its cytoplasm and nucleus are disintegrated and crescent vacant microspore presents; nutritive substances can't be accumulated at the stage of vacuolated microspore, cytoplasm is disintegrated, and microspore turns into a big vacant pollen. The ratio of abnormal development of male cells of SP3 is as high as 50%. This maybe relates to the treatment of space radiation, which results in chromosomal aberration, and also to the segregation and recombinatiom of chromosome of SP1 and SP2

  2. Resolving Tumor Heterogeneity: Genes Involved in Chordoma Cell Development Identified by Low-Template Analysis of Morphologically Distinct Cells

    Science.gov (United States)

    Wagner, Karin; Meditz, Katharina; Kolb, Dagmar; Feichtinger, Julia; Thallinger, Gerhard G.; Quehenberger, Franz; Liegl-Atzwanger, Bernadette; Rinner, Beate

    2014-01-01

    The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase ALG11 (695-fold) and the phosphatase subunit PPP2CB (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology. PMID:24503940

  3. Resolving tumor heterogeneity: genes involved in chordoma cell development identified by low-template analysis of morphologically distinct cells.

    Directory of Open Access Journals (Sweden)

    Amin El-Heliebi

    Full Text Available The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. UCHL3, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold and U-CH1 (3.7-fold cells. The mannosyltransferase ALG11 (695-fold and the phosphatase subunit PPP2CB (18.6-fold were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. TMEM144, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.

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

    Science.gov (United States)

    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. PMID:26285810

  5. Ascl3 knockout and cell ablation models reveal complexity of salivary gland maintenance and regeneration.

    Science.gov (United States)

    Arany, Szilvia; Catalán, Marcelo A; Roztocil, Elisa; Ovitt, Catherine E

    2011-05-15

    Expression of the transcription factor, Ascl3, marks a population of adult progenitor cells, which can give rise to both acinar and duct cell types in the murine salivary glands. Using a previously reported Ascl3(EGFP-Cre/+) knock-in strain, we demonstrate that Ascl3-expressing cells represent a molecularly distinct, and proliferating population of progenitor cells located in salivary gland ducts. To investigate both the role of the Ascl3 transcription factor, and the role of the cells in which it is expressed, we generated knockout and cell-specific ablation models. Ascl3 knockout mice develop smaller salivary glands than wild type littermates, but secrete saliva normally. They display a lower level of cell proliferation, consistent with their smaller size. In the absence of Ascl3, the cells maintain their progenitor function and continue to generate both acinar and duct cells. To directly test the role of the progenitor cells, themselves, in salivary gland development and regeneration, we used Cre-activated expression of diphtheria toxin (DTA) in the Ascl3-expressing (Ascl3+) cell population, resulting in specific cell ablation of Ascl3+ cells. In the absence of the Ascl3+ progenitor cells, the mice developed morphologically normal, albeit smaller, salivary glands able to secrete saliva. Furthermore, in a ductal ligation model of salivary gland injury, the glands of these mice were able to regenerate acinar cells. Our results indicate that Ascl3+ cells are active proliferating progenitors, but they are not the only precursors for salivary gland development or regeneration. We conclude that maintenance of tissue homeostasis in the salivary gland must involve more than one progenitor cell population.

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

    Science.gov (United States)

    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.

  7. Formation of the tetraploid intermediate is associated with the development of cells with more than four centrioles in the elastase-simian virus 40 tumor antigen transgenic mouse model of pancreatic cancer.

    OpenAIRE

    1991-01-01

    The development of pancreatic cancer in transgenic mice expressing the simian virus 40 tumor antigen placed under controlling regions of the elastase I gene is characterized by the sequential appearance of tetraploid and then multiple aneuploid cell populations. Pancreatic tissues from such transgenic mice were studied between 8 and 32 days of age. Virtually 100% of acinar cell nuclei had immunohistochemically detectable tumor antigen by 18 days. Tetraploid cells were demonstrated by DNA cont...

  8. In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

    Science.gov (United States)

    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

  9. The Regulatory Role of Rolipram on Inflammatory Mediators and Cholinergic/Adrenergic Stimulation-Induced Signals in Isolated Primary Mouse Submandibular Gland Cells

    Directory of Open Access Journals (Sweden)

    Dong Un Lee

    2016-01-01

    Full Text Available Exposure to bacterial lipopolysaccharides (LPS induces inflammatory signals in salivary glands. We investigated the regulatory role of phosphodiesterase 4 (PDE4 inhibitor rolipram on inflammatory mediators and cholinergic/adrenergic stimulation-induced intracellular Ca2+ signaling in salivary acinar and ductal cells. Submandibular gland (SMG expressed PDE4A through 4D mRNA and PDE4 was localized in the luminal membrane of SMG. LPS induced Ca2+ signaling and ROS production in SMG. Treatment with rolipram blocked LPS-induced Ca2+ increase and ROS production. The application of histamine evoked Ca2+ signals and ROS production, which were attenuated by rolipram in SMG cells. Moreover, LPS-induced NLRP3 inflammasome and cleaved caspase-1 were inhibited by rolipram. The inhibitory role of rolipram in ROS-induced Ca2+ signaling was mainly observed in acinar cells and not in ductal cells. Rolipram also protected SMG acinar but not ductal cells from LPS-induced cell membrane damage. In the case of cholinergic/adrenergic stimulation, carbachol/isoproterenol-induced Ca2+ signals were upregulated by the treatment of rolipram in SMG. In the case of cAMP-dependent ductal bicarbonate secretion by rolipram, no effect was observed on the modulation of ductal chloride/bicarbonate exchange activity. Rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against LPS-induced inflammation to protect the salivary gland cells.

  10. The Regulatory Role of Rolipram on Inflammatory Mediators and Cholinergic/Adrenergic Stimulation-Induced Signals in Isolated Primary Mouse Submandibular Gland Cells

    Science.gov (United States)

    Lee, Dong Un; Shin, Dong Min; Hong, Jeong Hee

    2016-01-01

    Exposure to bacterial lipopolysaccharides (LPS) induces inflammatory signals in salivary glands. We investigated the regulatory role of phosphodiesterase 4 (PDE4) inhibitor rolipram on inflammatory mediators and cholinergic/adrenergic stimulation-induced intracellular Ca2+ signaling in salivary acinar and ductal cells. Submandibular gland (SMG) expressed PDE4A through 4D mRNA and PDE4 was localized in the luminal membrane of SMG. LPS induced Ca2+ signaling and ROS production in SMG. Treatment with rolipram blocked LPS-induced Ca2+ increase and ROS production. The application of histamine evoked Ca2+ signals and ROS production, which were attenuated by rolipram in SMG cells. Moreover, LPS-induced NLRP3 inflammasome and cleaved caspase-1 were inhibited by rolipram. The inhibitory role of rolipram in ROS-induced Ca2+ signaling was mainly observed in acinar cells and not in ductal cells. Rolipram also protected SMG acinar but not ductal cells from LPS-induced cell membrane damage. In the case of cholinergic/adrenergic stimulation, carbachol/isoproterenol-induced Ca2+ signals were upregulated by the treatment of rolipram in SMG. In the case of cAMP-dependent ductal bicarbonate secretion by rolipram, no effect was observed on the modulation of ductal chloride/bicarbonate exchange activity. Rolipram could suppress the inflammatory signals and could be a potential therapeutic strategy against LPS-induced inflammation to protect the salivary gland cells. PMID:27143817

  11. Cryptosporidium Lactate Dehydrogenase Is Associated with the Parasitophorous Vacuole Membrane and Is a Potential Target for Developing Therapeutics.

    Science.gov (United States)

    Zhang, Haili; Guo, Fengguang; Zhu, Guan

    2015-01-01

    The apicomplexan, Cryptosporidium parvum, possesses a bacterial-type lactate dehydrogenase (CpLDH). This is considered to be an essential enzyme, as this parasite lacks the Krebs cycle and cytochrome-based respiration, and mainly-if not solely, relies on glycolysis to produce ATP. Here, we provide evidence that in extracellular parasites (e.g., sporozoites and merozoites), CpLDH is localized in the cytosol. However, it becomes associated with the parasitophorous vacuole membrane (PVM) during the intracellular developmental stages, suggesting involvement of the PVM in parasite energy metabolism. We characterized the biochemical features of CpLDH and observed that, at lower micromolar levels, the LDH inhibitors gossypol and FX11 could inhibit both CpLDH activity (Ki = 14.8 μM and 55.6 μM, respectively), as well as parasite growth in vitro (IC50 = 11.8 μM and 39.5 μM, respectively). These observations not only reveal a new function for the poorly understood PVM structure in hosting the intracellular development of C. parvum, but also suggest LDH as a potential target for developing therapeutics against this opportunistic pathogen, for which fully effective treatments are not yet available. PMID:26562790

  12. MavN is a Legionella pneumophila vacuole-associated protein required for efficient iron acquisition during intracellular growth

    Science.gov (United States)

    Isaac, Dervla T.; Laguna, Rita K.; Valtz, Nicole; Isberg, Ralph R.

    2015-01-01

    Iron is essential for the growth and virulence of most intravacuolar pathogens. The mechanisms by which microbes bypass host iron restriction to gain access to this metal across the host vacuolar membrane are poorly characterized. In this work, we identify a unique intracellular iron acquisition strategy used by Legionella pneumophila. The bacterial Icm/Dot (intracellular multiplication/defect in organelle trafficking) type IV secretion system targets the bacterial-derived MavN (more regions allowing vacuolar colocalization N) protein to the surface of the Legionella-containing vacuole where this putative transmembrane protein facilitates intravacuolar iron acquisition. The ΔmavN mutant exhibits a transcriptional iron-starvation signature before its growth is arrested during the very early stages of macrophage infection. This intracellular growth defect is rescued only by the addition of excess exogenous iron to the culture medium and not a variety of other metals. Consistent with MavN being a translocated substrate that plays an exclusive role during intracellular growth, the mutant shows no defect for growth in broth culture, even under severe iron-limiting conditions. Putative iron-binding residues within the MavN protein were identified, and point mutations in these residues resulted in defects specific for intracellular growth that are indistinguishable from the ΔmavN mutant. This model of a bacterial protein inserting into host membranes to mediate iron transport provides a paradigm for how intravacuolar pathogens can use virulence-associated secretion systems to manipulate and acquire host iron. PMID:26330609

  13. Ultracytochemical localization of H+—adenosine triphosphatase activity in autophagic vacuoles induced by vinblastine in rat liver

    Institute of Scientific and Technical Information of China (English)

    LUOSHENQIU; MASAHIROSAKAI; 等

    1990-01-01

    H-adenosine triphosphatase (H+-ATPase) activity was demonstrated cytochemically in autophagic vacuoles(AVs) of rat hepatocytes using a modification of the method for the demonstration of neutral p-nitrophenyl phosphatase(p-NPPase) activity[1].When an inhibitor of H+-ATPase,N-ethylmaleimide (NEM) or 4,4'-diisothiocyanostilbene-2,2'disulfonic acid,disodium salt (DIDS) was included in the incubation medium the enyzme activity was abolished indicating that p-NPPase demonstrated in this study represents H+-ATPase.Autophagy was induced by a single intraperitoneal injection of vinblastine sulfate(VBL).The number of AVs increased remarkably in hepatocytes from 40 min after VBL treatment.H+-ATPase activity was observed mainly on the membranes of lysosomes and AVs.However,early forms of AVs containing only incompletely digested material showed no H+-ATPase activity.Most AVs revealing a positive reaction seemed to be in advanced stages of development.Acid phosphatase acticity was demonstrable in mature but not in early forms of AVs.The present investigation showed that membranes of advanced stage AVs possess an H+-ATPase which may be derived from lysosomal membranes.

  14. Morphology of the pancreas of some species belonging to the genera Phelsuma and Gecko (family Gekkonidae): evidence of apoptotic process during the seasonal cycle.

    Science.gov (United States)

    Buono, S; Odierna, G; Putti, R

    2006-10-01

    In this study we investigated comparative morphology of the endocrine pancreas of several species belonging to the family Gekkonidae and apoptotic processes of the pancreas which may be correlated to the seasonal cycle. The following species of the family Gekkonidae were studied: Phelsuma lineata, P. madagascariensis, P. dubia, P. abotti, Gekko gecko, G. vittatus, and Geckonia chazaliae. In all these species the pancreas consisted of large and medium islets as well as endocrine cells which were scattered throughout the acinar cells. Exocrine parenchyma consisted of tubuli-acini. Four mayor cell types were identified in the endocrine pancreas, using immunocytochemistry: glucagon-immunoreactive (A) cells, insulin-immunoreactive (B) cells, somatostatin-immunoreactive (D) cells, and pancreatic polypeptide immunoreactive (PP) cells. In the endocrine pancreas the amount of A cells and B cells was either equal or a prevalence of A cells was observed. In the wet season the pancreatic morphology presented normal features with very rare apoptotic cells. The animals belonging to the genus Phelsuma taken in the dry season (July) showed numerous vacuolated, Caspase 3, 9 and 11-immunoreactive acinar and some endocrine cells containing picnotic nuclei which were positive to tunel reaction. The animals belonging to the genus Gekko taken at the end of the dry season (October) exhibited strongly vacuolated, Caspase 3, 9 and 11-immunoreactive endocrine and some acinar cells containing nuclei which were positive to tunel reaction. These apoptosis events could be a reaction in response to stress mechanisms, such as a starvation period during the dry season. PMID:16763810

  15. Salt stress induces internalization of plasma membrane aquaporin into the vacuole in Arabidopsis thaliana.

    Science.gov (United States)

    Ueda, Masamichi; Tsutsumi, Nobuhiro; Fujimoto, Masaru

    2016-06-10

    Salt stress is a major environmental stress for plants, causing hyperosmotic, ionic and drought-like stresses. Plasma membrane intrinsic protein 2;1 (PIP2;1), which forms a water channel that regulates water flux thorough the plasma membrane (PM), is constitutively trafficked between the PM and the trans-Golgi network (TGN) in Arabidopsis thaliana. Salt stress is known to relocalize PIP2;1 to intracellular compartments, probably to decrease the water permeability of the root. However, the destination of internalized PIP2;1 and the mechanism by which PIP2;1 is internalized remain unclear. Here, we examined the effects of salt stress and inhibitors of endocytosis on the intracellular localization of green fluorescent protein-fused PIP2;1 (GFP-PIP2;1) in Arabidopsis thaliana root epidermal cells. Salt stress decreased the fluorescence of GFP-PIP2;1 at the PM and increased it in the vacuolar lumen as shown by staining of the vacuolar membrane. The internalization of PIP2;1 was suppressed by an inhibitor of clathrin-mediated endocytosis and by inhibitors of two kinases that appear to have roles in salt stress, phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 4-kinase (PI4K). Inhibiting PI4K suppressed the salt-induced endocytosis of GFP-PIP2;1 at the PM, whereas inhibiting PI3K suppressed the trafficking of GFP-PIP2;1 after its internalization. These results suggest that salt stress induces the internalization of PIP2;1 from the PM to the vacuolar lumen, and that these processes are dependent on clathrin, PI3K and PI4K. PMID:27163638

  16. Autonomous isolation, long-term culture and differentiation potential of adult salivary gland-derived stem/progenitor cells.

    Science.gov (United States)

    Baek, Hyunjung; Noh, Yoo Hun; Lee, Joo Hee; Yeon, Soo-In; Jeong, Jaemin; Kwon, Heechung

    2014-09-01

    Salivary gland stem/progenitor cells belong to the endodermal lineage and may serve as good candidates to replace their dysfunctional counterparts. The objective of this study was to isolate large numbers of salivary gland tissue-derived stem cells (SGSCs) from adult rats in order to develop a clinically applicable method that does not involve sorting or stem cell induction by duct ligation. We analysed SGSCs isolated from normal rat salivary glands to determine whether they retained the major characteristics of stem cells, self-renewal and multipotency, especially with respect to the various endodermal cell types. SGSCs expressed high levels of integrin α6β1 and c-kit, which are surface markers of SGSCs. In particular, the integrin α6β1(+) /c-kit(+) salivary gland cells maintained the morphology, proliferation activity and multipotency of stem cells for up to 92 passages in 12 months. Furthermore, we analysed the capacity of SGSCs to differentiate into endoderm lineage cell types, such as acinar-like and insulin-secreting cells. When cultured on growth factor reduced matrigel, the morphology of progenitor cells changed to acinar-like structures and these cells expressed the acinar cell-specific marker, α-amylase, and tight junction markers. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) data showed increased expression of pancreatic cell markers, including insulin, Pdx1, pan polypeptide and neurogenin-3, when these cells formed pancreatic clusters in the presence of activin A, exendin-4 and retinoic acid. These data demonstrate that adult salivary stem/progenitor cells may serve as a potential source for cell therapy in salivary gland hypofunction and diabetes.

  17. The Measurement of Membrane Potential and NO3 Activity in Root Cells Using Ion-Selective Microelectrodes

    Institute of Scientific and Technical Information of China (English)

    FAN Xiao-rong; Anthony J Miller; SHEN Qi-rong

    2003-01-01

    Remobilisation of nitrate in plants, especially in vacuole of plant, is mostly related to the qua-lity of agricultural products and the high nitrogen use efficiency in plants. Ion-selective microelectrodes offer anon-destructive and non-interruptive method to measure NO-3 gradients and electric potential differences acrossboth the plasma membrane and tonoplast. Thus, a double-barrelled microelectrode backfilled with a mem-brane sensor for NO-3 embedded in poly vinyl chloride (PVC) can record the NO-3 activity in cytoplasm and vac-uole of a cell. This paper presented how to make this kind of microelectrode and how to do the intracellularmeasurements on intact plants. Our result showed that nitrate activity was about 2.7 mmol L-1 in cytoplasmwhile 70 mmol L-1 in vacuole, which implicated that vacuole was a pool of nitrate in plants.

  18. Cell Secretion: Current Structural and Biochemical Insights

    Directory of Open Access Journals (Sweden)

    Saurabh Trikha

    2010-01-01

    Full Text Available Essential physiological functions in eukaryotic cells, such as release of hormones and digestive enzymes, neurotransmission, and intercellular signaling, are all achieved by cell secretion. In regulated (calcium-dependent secretion, membrane-bound secretory vesicles dock and transiently fuse with specialized, permanent, plasma membrane structures, called porosomes or fusion pores. Porosomes are supramolecular, cup-shaped lipoprotein structures at the cell plasma membrane that mediate and control the release of vesicle cargo to the outside of the cell. The sizes of porosomes range from 150nm in diameter in acinar cells of the exocrine pancreas to 12nm in neurons. In recent years, significant progress has been made in our understanding of the porosome and the cellular activities required for cell secretion, such as membrane fusion and swelling of secretory vesicles. The discovery of the porosome complex and the molecular mechanism of cell secretion are summarized in this article.

  19. Sensitivity of Hep G2 cells to Bacillus cereus emetic toxin.

    Science.gov (United States)

    Kamata, Yoichi; Kanno, Shinji; Mizutani, Noriko; Agata, Norio; Kawakami, Hiroshi; Sugiyama, Kei-ichi; Sugita-Konishi, Yoshiko

    2012-11-01

    We herein examined the sensitivity of Hep G2 human hepatoma cells to Bacillus cereus emetic toxin. Hep G2 cells were treated with the emetic toxin, and the cell shape was observed. The same experiments were performed for comparison purposes, using HEp-2 cells, which are currently used by most laboratories for a bioassay of the emetic toxin. Hep G2 cells showed clearer vacuolation in the cytosol within 2 hr and required a shorter incubation period than HEp-2 cells (10 hr). The number of vacuoles in the Hep G2 cells was greater, and the size of the vacuoles was larger than those observed in HEp-2 cells. The minimal concentration of the emetic toxin required to induce the vacuolation of Hep G2 cells was 0.04 ng/ml. The concentration for the HEp-2 cells was 1 ng/ml. These findings indicate that Hep G2 cells show higher sensitivity to the emetic toxin. Hep G2 cells may be superior to the currently used HEp-2 cells for the bioassay of the emetic toxin.

  20. Rescue of salivary gland function after stem cell transplantation in irradiated glands.

    Directory of Open Access Journals (Sweden)

    Isabelle M A Lombaert

    Full Text Available Head and neck cancer is the fifth most common malignancy and accounts for 3% of all new cancer cases each year. Despite relatively high survival rates, the quality of life of these patients is severely compromised because of radiation-induced impairment of salivary gland function and consequential xerostomia (dry mouth syndrome. In this study, a clinically applicable method for the restoration of radiation-impaired salivary gland function using salivary gland stem cell transplantation was developed. Salivary gland cells were isolated from murine submandibular glands and cultured in vitro as salispheres, which contained cells expressing the stem cell markers Sca-1, c-Kit and Musashi-1. In vitro, the cells differentiated into salivary gland duct cells and mucin and amylase producing acinar cells. Stem cell enrichment was performed by flow cytrometric selection using c-Kit as a marker. In vitro, the cells differentiated into amylase producing acinar cells. In vivo, intra-glandular transplantation of a small number of c-Kit(+ cells resulted in long-term restoration of salivary gland morphology and function. Moreover, donor-derived stem cells could be isolated from primary recipients, cultured as secondary spheres and after re-transplantation ameliorate radiation damage. Our approach is the first proof for the potential use of stem cell transplantation to functionally rescue salivary gland deficiency.

  1. Ultrastructural and cytochemical aspects of the basophilic cells in the hepatopancreas ofAplysia depilans(Mollusca, Opisthobranchia).

    Science.gov (United States)

    Lobo-da-Cunha, A

    1999-02-01

    The basophilic cells ofAplysia depilanshave a pyramidal shape and a large nucleus usually located near the center or in the basal half of the cell. The nucleus possesses several clumps of condensed chromatin and a prominent nucleolus. The great profusion of rough endoplasmic reticulum cisterns in a major feature of these cells. Secretion granules are accumulated in the apical zone, and arylsulphatase was detected in some of them. In some basophilic cells a very substantial part of the cell volume was occupied by clear vacuoles, some of them reaching 9 mum. However, in other cells only a few vacuoles were observed. Probably the cells with just a few vacuoles are still young, and after a progressive accumulation, the vacuoles become abundant in old cells. The presence of a dark nucleus in the cells with a large number of vacuoles suggests that they are in a final stage of their life. Arylsulphatase was detected in the vacuoles and also in small secondary lysosomes containing substances in digestion. Bundles of tubules with 50 nm in diameter were found within some cisterns of rough endoplasmic reticulum. A cell fraction enriched in mannitol oxidase, extracted from the hepatopancreas of a terrestrial slug, consisted in very similar tubular structures. Using a histochemical method, mannitol oxidase was detected in the basophilic cells ofA. depilans, and it may be associated with the tubular structures of the endoplasmic reticulum. This is the first report of mannitol oxidase in opisthobranch molluscs. Almost spherical peroxisomes with a small nucleoid were abundant in these cells. The nucleoids presented a rectangular section, but a crystalline structure was not evident. The peroxisomes were stained after the cytochemical detection of catalase activity. PMID:18627851

  2. Mechanisms Underlying Activation of α1-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions

    Science.gov (United States)

    Bragiel, Aneta M.; Wang, Di; Pieczonka, Tomasz D.; Shono, Masayuki; Ishikawa, Yasuko

    2016-01-01

    Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α1-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La3+, implying the participation of store-operated Ca2+ channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La3+, suggesting the involvement of extracellular Ca2+ entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca2+/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca2+ entry. PMID:27367668

  3. Analysis of the clinical pathologic acinar cell carcinoma of salivary gland%涎腺腺泡细胞癌临床病理分析

    Institute of Scientific and Technical Information of China (English)

    林楚忠; 吴小霞; 李广文; 苏文雄

    2014-01-01

    目的 探讨涎腺腺泡细胞癌的临床特点、组织形态学特征、免疫组化特点、诊断、治疗及预后.方法 复习15例涎腺腺泡细胞癌手术切除标本的病理切片,并进行免疫组织化学染色,结合相关临床资料进行分析.结果 涎腺腺泡细胞癌各年龄段均可发病,好发于腮腺,生长缓慢,病程长,预后相对较好.涎腺腺泡细胞癌可见包膜,大部分肿物界限清楚,小部分肿物与周围组织粘连较紧,边界不清.组织形态学可见肿瘤细胞呈圆形,胞浆丰富,嗜碱性或透明,细胞核较小、深染,无明显异型性,排列成实性片状及腺泡状.免疫组织化学显示:CK阳性、S100部分病例阳性,β-catenin在73.3%的病例中存在异常表达,Ki-67阳性指数为5%~30%.结论 涎腺腺泡细胞癌发病少,属低度恶性肿瘤,影像学诊断对其无特异性,主要依靠病理诊断来证实,治疗以根治性手术切除为主,术后可辅以放射治疗,以减少肿瘤复发及转移.

  4. Enhanced proliferation of acinar and progenitor cells by prophylactic pilocarpine treatment underlies the observed amelioration of radiation injury to parotid glands

    NARCIS (Netherlands)

    Burlage, Fred R.; Faber, Hette; Kampinga, Harm H.; Langendijk, Johannes A.; Vissink, Arjan; Coppes, Rob P.

    2009-01-01

    Background: Administration of pilocarpine before irradiation can ameliorate radiation-induced hyposalivation. Indirect evidence Suggests that this effect may be mediated through induction of a compensatory response. In this study, this hypothesis is tested directly, by assessing the proliferation of

  5. Membrane potential and conductance of frog skin gland acinar cells in resting conditions and during stimulation with agonists of macroscopic secretion

    DEFF Research Database (Denmark)

    Sørensen, Jakob B.; Larsen, Erik Hviid

    1999-01-01

    Adrenaline; carbachol; Cl- secretion; exocrine gland; isoproterenol; noradrenaline; prostaglandin E*U2......Adrenaline; carbachol; Cl- secretion; exocrine gland; isoproterenol; noradrenaline; prostaglandin E*U2...

  6. Symbiotic Chlorella variabilis incubated under constant dark conditions for 24 hours loses the ability to avoid digestion by host lysosomal enzymes in digestive vacuoles of host ciliate Paramecium bursaria.

    Science.gov (United States)

    Kodama, Yuuki; Fujishima, Masahiro

    2014-12-01

    Endosymbiosis between symbiotic Chlorella and alga-free Paramecium bursaria cells can be induced by mixing them. To establish the endosymbiosis, algae must acquire temporary resistance to the host lysosomal enzymes in the digestive vacuoles (DVs). When symbiotic algae isolated from the alga-bearing paramecia are kept under a constant dark conditions for 24 h before mixing with the alga-free paramecia, almost all algae are digested in the host DVs. To examine the cause of algal acquisition to the host lysosomal enzymes, the isolated algae were kept under a constant light conditions with or without a photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea for 24 h, and were mixed with alga-free paramecia. Unexpectedly, most of the algae were not digested in the DVs irrespective of the presence of the inhibitor. Addition of 1 mM maltose, a main photosynthetic product of the symbiotic algae or of a supernatant of the isolated algae kept for 24 h under a constant light conditions, did not rescue the algal digestion in the DVs. These observations reveal that unknown factors induced by light are a prerequisite for algal resistance to the host lysosomal enzymes.

  7. Subcellular localization of Cd in the root cells of Allium sativum by electron energy loss spectroscopy

    Indian Academy of Sciences (India)

    Donghua Liu; Ingrid Kottke

    2003-06-01

    The ultrastructural investigation of the root cells of Allium sativum L. exposed to three different concentrations of Cd (100 M, 1 mM and 10 mM) for 9 days was carried out. The results showed that Cd induced several significant ultrastructural changes – high vacuolization in cytoplasm, deposition of electron-dense material in vacuoles and nucleoli and increment of disintegrated organelles. Data from electron energy loss spectroscopy (EELS) revealed that Cd was localized in the electron-dense precipitates in the root cells treated with 10 mM Cd. High amounts of Cd were mainly accumulated in the vacuoles and nucleoli of cortical cells in differentiating and mature root tissues. The mechanisms of detoxification and tolerance of Cd are briefly explained.

  8. Lung adenocarcinoma with clear cell features producing carbohydrate antigen 19-9.

    Science.gov (United States)

    Goto, Taichiro; Hada, Masao; Oyama, Toshio

    2015-10-01

    A 76-year-old man underwent surgery for lung cancer. Histopathologically, most of the resected tumor was composed of polygonal cells with foamy cytoplasm, and the cells were arranged predominantly in acinar patterns. In this case, although the carbohydrate antigen 19-9 level was high before surgery, it normalized after resection. The tumor was considered a carbohydrate antigen 19-9-producing tumor, which was further supported by the results of immunohistochemical analysis. Adenocarcinoma with clear cell features, producing carbohydrate antigen 19-9, is an exceedingly rare entity.

  9. 1-Nitropyrene (1-NP) induces apoptosis and apparently a non-apoptotic programmed cell death (paraptosis) in Hepa1c1c7 cells

    International Nuclear Information System (INIS)

    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 that 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

  10. Proliferation and Differentiation of Duct Epithelial Cells after Partial Pancreatectomy in Rats

    Institute of Scientific and Technical Information of China (English)

    LIU Tao; WANG Chunyou; WAN Chidan; XIONG Jiongxin; ZHOU Feng

    2006-01-01

    The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5.There was significant difference between the experimental group and the control group (P<0.001).Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.

  11. Myoepithelial cells: Current perspectives in salivary gland tumors

    Directory of Open Access Journals (Sweden)

    C Pramod Redder

    2013-01-01

    Full Text Available Myoepithelial cells are normal constituent of the salivary acini and smaller ducts, and are found between the epithelial cells and the basement membrane. Microscopic examination shows that myoepithelial cells are thin and spindle-shaped and situated between the basement membrane and epithelial cells. Ultrastructurally they possess a number of cytoplasmic processes that extend between and over the acinar and ductal-lining cells. They display features of both smooth muscle and epithelium, such as numerous microfilaments with focal densities in the cytoplasmic processes, and desmosomes which attach the myoepithelial to the epithelial cells. Neoplastic myoepithelial cells in both benign and malignant tumors can take several forms, including epithelioid, spindle, plasmacytoid, and clear, and this variability largely accounts for difficulties in histopathological diagnosis. This review article highlights the role of myoepithelial cells in salivary gland tumors.

  12. Endocytic Trafficking towards the Vacuole Plays a Key Role in the Auxin Receptor SCFTIR-Independent Mechanism of Lateral Root Formation in A.thaliana

    Institute of Scientific and Technical Information of China (English)

    Patricio Pérez-Henríquez; Natasha V.Raikhel; Lorena Norambuena

    2012-01-01

    Plants' developmental plasticity plays a pivotal role in responding to environmental conditions.One of the most plastic plant organs is the root system.Different environmental stimuli such as nutrients and water deficiency may induce lateral root formation to compensate for a low level of water and/or nutrients.It has been shown that the hormone auxin tunes lateral root development and components for its signaling pathway have been identified.Using chemical biology,we discovered an Arabidopsis thaliana lateral root formation mechanism that is independent of the auxin receptor SCFTIR.The bioactive compound Sortin2 increased lateral root occurrence by acting upstream from the morphological marker of lateral root primordium formation,the mitotic activity.The compound did not display auxin activity.At the cellular level,Sortin2 accelerated endosomal trafficking,resulting in increased trafficking of plasma membrane recycling proteins to the vacuole.Sortin2 affected Late endosome/PVC/MVB trafficking and morphology.Combining Sortin2 with well-known drugs showed that endocytic trafficking of Late E/PVC/MVB towards the vacuole is pivotal for Sortin2induced SCFTIR-independent lateral root initiation.Our results revealed a distinctive role for endosomal trafficking in the promotion of lateral root formation via a process that does not rely on the auxin receptor complex SCFTIR.

  13. Vacuolating cytotoxin genotypes are strong markers of gastric cancer and duodenal ulcer-associated Helicobacter pylori strains: a matched case-control study.

    Science.gov (United States)

    Memon, Ameer A; Hussein, Nawfal R; Miendje Deyi, Véronique Y; Burette, Alain; Atherton, John C

    2014-08-01

    The Helicobacter pylori virulence gene, cagA, and active forms of the vacuolating cytotoxin gene, vacA, are major determinants of pathogenesis. However, previous studies linking these factors to disease risk have often included patients using aspirin/nonsteroidal anti-inflammatory agents (NSAIDs) or acid-suppressing drugs, both of which may confound results. Also, particularly for gastric cancer (GC), controls have often been of quite different ages. Here, we performed a careful study in a "clean" Belgian population with gastric cancer cases age and sex matched to 4 controls and with a parallel duodenal ulcer (DU) group. As in other populations, there was a close association between the presence of cagA and the vacA s1 genotype. For GC, associations were found for vacA s1-positive (P = 0.01, odds ratio [OR], 9.37; 95% confidence interval [CI], 1.16 to 201.89), i1-positive (P = 0.003; OR, 12.08; 95% CI, 1.50 to 259.64), and cagA-positive status (P ulcer-associated strains are the vacA s1 and i1 genotypes. This fits with experimental data showing that the s and i regions are the key determinants of vacuolating cytotoxin activity.

  14. Effects of Estrogen and/or Progesterone on Morphology of Pituitary Luteinizing Hormone Cells

    Institute of Scientific and Technical Information of China (English)

    周寿康; 任惠民; 王健; 赵伟; 顾敦瑜; 谢衷明

    2001-01-01

    Objective To identify the morphological characteristics of pituitary luteinizing hormone (LH) cells after exogenous gonadal hormone treatment Methods Effects of various doses of estrogen, progesterone and their combination on morphological parameters, including the size and shape of pituitary LH cells, the size of endocellular vacuoles, were observed and measured by immuno-histochemistry and computer image analysis.Results Different kinds of gonadal hormones could recover the magnified LH cells to the normal level in ovariectomized rats. However, their final effects on the gonadotrophin levels and the cellular morphological characters of the LH cells were different. The low dose of estrogen elicited abundant hormone stored in the LH cells to an easy released status with a lot of different size of vacuoles. On the contrary, the high dose of estrogen inhibited the storage of LH, and the LH cells were filled with secretory granules and few vacuoles. The progesterone could promote the storage of LH in an uneasy released status. The administration of estrogen-progesterone combina tion not only inhibited the storage of LH, but also the release of LH. In this group,the LH cells containing a large amount of secretory granules and a few vacuoles showed a better uniform shape compared with those administrated with high dose of estrogen.Conclusion: Different kinds of gonadal hormones could reverse the excessive secretion of LH and recover the morphological change of LH cells to the normally physiological condition.

  15. Creating new β cells: cellular transmutation by genomic alchemy.

    Science.gov (United States)

    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. PMID:23434598

  16. Lack of robust satellite cell activation and muscle regeneration during the progression of Pompe disease

    OpenAIRE

    Schaaf, Gerben J.; van Gestel, Tom JM; Brusse, Esther; Verdijk, Robert M.; de Coo, Irenaeus FM; Doorn Van, Pieter A; Ploeg, Ans T van der; Pijnappel, WWM Pim

    2015-01-01

    Introduction Muscle stem cells termed satellite cells are essential for muscle regeneration. A central question in many neuromuscular disorders is why satellite cells are unable to prevent progressive muscle wasting. We have analyzed muscle fiber pathology and the satellite cell response in Pompe disease, a metabolic myopathy caused by acid alpha-glucosidase deficiency and lysosomal glycogen accumulation. Pathology included muscle fiber vacuolization, loss of cross striation, and immune cell ...

  17. [Effect of stress conditions on the activity and isozyme composition of peroxidase of vacuoles and tissue extract of red beet roots].

    Science.gov (United States)

    Pradedova, E V; Nimaeva, O D; Saliaev, R K

    2014-01-01

    Changes in the enzymatic activity of phenol-dependent peroxidase (PO) of vacuoles and tissue extract of red beet (Beta vulgaris L.) roots in different phases of plant development and in hyperosmotic stress and pathogen infection were found. The highest activity was observed during root growth and the lowest PO activity occurred in dormancy, respectively. Activation of the enzyme was observed in infected roots. The isozyme composition of PO was characterized by lability, and the number of cationic isoforms varied significantly. The optimum pH of the enzyme changed depending on the growth phase and stressor, tending to shift towards low values at rest and in hyperosmotic stress. The shift in the optimum pH coincided with the appearance of additional cationic PO isoforms. PMID:25731036

  18. Mechanisms of mouse spleen dendritic cell function in the generation of influenza-specific, cytolytic T lymphocytes

    OpenAIRE

    1992-01-01

    We have evaluated the capacity of dendritic cells to function as antigen-presenting cells (APCs) for influenza and have examined their mechanism of action. Virus-pulsed dendritic cells were 100 times more efficient than bulk spleen cells in stimulating cytotoxic T lymphocyte (CTL) formation. The induction of CTLs required neither exogenous lymphokines nor APCs in the responding T cell population. Infectious virus entered dendritic cells through intracellular acidic vacuoles and directed the s...

  19. Clear cell renal cell carcinoma with hemangioblastoma-like features: A recently described pattern with unusual immunohistochemical profile

    Directory of Open Access Journals (Sweden)

    Sankalp Sancheti

    2015-01-01

    Full Text Available The diagnosis of clear cell renal cell carcinoma may sometimes pose challenges because of the presence of uncharacteristic morphology, varied immunophenotypic patterns and due to lack of molecular or genetic determinants. More often, the morphological variations can be easily overlooked in routine practice and a more common diagnosis is usually put forward. Solid, acinar and alveolar are the common patterns described in the literature. We report a recently described pattern of clear cell renal cell carcinoma which has hemangioblastoma-like morphology and an unusual immunoprofile. In our case, the tumor showed a diffuse hemangioblastoma-like pattern and diffuse positivity for Alpha-inhibin on immunohistochemistry. A thorough literature search, extensive sampling and an expanded immunohistochemistry panel revealed a clear cell renal cell carcinoma component. Presence of renal vein thrombosis and focal necrosis were other helpful features in discerning the malignant nature of tumor.

  20. Internalization of cycloheptaamylose-dansyl chloride complex during labelling of surface membrane in living Paramecium aurelia cells.

    Science.gov (United States)

    Giordano, P A; Wyroba, E; Bottiroli, G

    1985-01-01

    Internalization of cycloheptaamylose-dansyl chloride complex during surface labelling of living long-term starved Paramecium aurelia cells has been observed. This process may be inhibited by pretreatment of the ciliates with dichloroisoproterenol. Uptake of cycloheptaamylose-dansyl chloride may be visualized only after UV preirradiation: the appearance of orange-fluorescing vacuoles of diameter 2.3-4.5 micron may then be observed. Microspectrographic analysis performed on the cells and dansyl derivatives indicates that this fluorescence is produced by a photochemical reaction of dansyl chloride - released from CDC complex inside the digestive vacuoles-under the influence of UV irradiation.

  1. Role of bone marrow-derived mesenchymal stem cells in a rat model of severe acute pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Xiao-Huang Tu; Jing-Xiang Song; Xiao-Jun Xue; Xian-Wei Guo; Yun-Xia Ma; Zhi-Yao Chen; Zhong-Dong Zou; Lie Wang

    2012-01-01

    AIM:To investigate the role and potential mechanisms of bone marrow mesenchymal stem cells (MSCs) in severe acute peritonitis (SAP).METHODS:Pancreatic acinar cells from Sprague Dawley rats were randomly divided into three groups:nonsodium deoxycholate (SDOC) group (non-SODC group),SDOC group,and a MSCs intervention group (i.e.,a co-culture system of MSCs and pancreatic acinar cells + SDOC).The cell survival rate,the concentration of malonaldehyde (MDA),the density of superoxide dismutase (SOD),serum amylase (AMS) secretion rate and lactate dehydrogenase (LDH) leakage rate were detected at various time points.In a separate study,Sprague Dawley rats were randomly divided into either an SAP group or an SAP + MSCs group.Serum AMS,MDA and SOD,interleukin (IL)-6,IL-10,and tumor necrosis factor (TNF)-α levels,intestinal mucosa injury scores and proliferating cells of small intestinal mucosa were measured at various time points after injecting either MSCs or saline into rats.In both studies,the protective effect of MSCs was evaluated.RESULTS:In vitro,The cell survival rate of pancreatic acinar cells and the density of SOD were significantly reduced,and the concentration of MDA,AMS secretion rate and LDH leakage rate were significantly increased in the SDOC group compared with the MSCs intervention group and the Non-SDOC group at each time point.In vivo,Serum AMS,IL-6,TNF-α and MAD level in the SAP + MSCs group were lower than the SAP group;however serum IL-10 level was higher than the SAP group.Serum SOD level was higher than the SAP group at each time point,whereas a significant betweengroup difference in SOD level was only noted after 24 h.Intestinal mucosa injury scores was significantly reduced and the proliferating cells of small intestinal mucosa became obvious after injecting MSCs.CONCLUSION:MSCs can effectively relieve injury to pancreatic acinar cells and small intestinal epithelium,promote the proliferation of enteric epithelium and repair of the mucosa

  2. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Bhanot Haymanti

    2011-06-01

    Full Text Available Abstract Background Methuosis is a unique form of non-apoptotic cell death triggered by alterations in the trafficking of clathrin-independent endosomes, ultimately leading to extreme vacuolization and rupture of the cell. Results Here we describe a novel chalcone-like molecule, 3-(2-methyl-1H- indol-3-yl-1-(4-pyridinyl-2-propen-1-one (MIPP that induces cell death with the hallmarks of methuosis. MIPP causes rapid accumulation of vacuoles derived from macropinosomes, based on time-lapse microscopy and labeling with extracellular fluid phase tracers. Vacuolization can be blocked by the cholesterol-interacting compound, filipin, consistent with the origin of the vacuoles from non-clathrin endocytic compartments. Although the vacuoles rapidly acquire some characteristics of late endosomes (Rab7, LAMP1, they remain distinct from lysosomal and autophagosomal compartments, suggestive of a block at the late endosome/lysosome boundary. MIPP appears to target steps in the endosomal trafficking pathway involving Rab5 and Rab7, as evidenced by changes in the activation states of these GTPases. These effects are specific, as other GTPases (Rac1, Arf6 are unaffected by the compound. Cells treated with MIPP lose viability within 2-3 days, but their nuclei show no evidence of apoptotic changes. Inhibition of caspase activity does not protect the cells, consistent with a non-apoptotic death mechanism. U251 glioblastoma cells selected for temozolomide resistance showed sensitivity to MIPP-induced methuosis that was comparable to the parental cell line. Conclusions MIPP might serve as a prototype for new drugs that could be used to induce non-apoptotic death in cancers that have become refractory to agents that work through DNA damage and apoptotic mechanisms.

  3. p53 mutations cooperate with oncogenic Kras to promote adenocarcinoma from pancreatic ductal cells.

    Science.gov (United States)

    Bailey, J M; Hendley, A M; Lafaro, K J; Pruski, M A; Jones, N C; Alsina, J; Younes, M; Maitra, A; McAllister, F; Iacobuzio-Donahue, C A; Leach, S D

    2016-08-11

    Pancreatic cancer is one of the most lethal malignancies, with virtually all patients eventually succumbing to their disease. Mutations in p53 have been documented in >50% of pancreatic cancers. Owing to the high incidence of p53 mutations in PanIN 3 lesions and pancreatic tumors, we interrogated the comparative ability of adult pancreatic acinar and ductal cells to respond to oncogenic Kras and mutant Tp53(R172H) using Hnf1b:CreER(T2) and Mist1:CreER(T2) mice. These studies involved co-activation of a membrane-tethered GFP lineage label, allowing for direct visualization and isolation of cells undergoing Kras and mutant p53 activation. Kras activation in Mist1(+) adult acinar cells resulted in brisk PanIN formation, whereas no evidence of pancreatic neoplasia was observed for up to 6 months following Kras activation in Hnf1beta(+) adult ductal cells. In contrast to the lack of response to oncogenic Kras alone, simultaneous activation of Kras and mutant p53 in adult ductal epithelium generated invasive PDAC in 75% of mice as early as 2.5 months after tamoxifen administration. These data demonstrate that pancreatic ductal cells, whereas exhibiting relative resistance to oncogenic Kras alone, can serve as an effective cell of origin for pancreatic ductal adenocarcinoma in the setting of gain-of-function mutations in p53. PMID:26592447

  4. Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy

    NARCIS (Netherlands)

    Tucker, Katherine A; Reggiori, Fulvio; Dunn, William A; Klionsky, Daniel J; Reggiori, Fulvio

    2003-01-01

    Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins. This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the deliv