scholarly journals Karyopherin Alpha 2-Expressing Pancreatic Duct Glands and Intra-Islet Ducts in Aged Diabetic C414A-Mutant-CRY1 Transgenic Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Satoshi Okano ◽  
Akira Yasui ◽  
Shin-ichiro Kanno ◽  
Kennichi Satoh ◽  
Masahiko Igarashi ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Pancreatic Duct ◽  
Zinc Binding ◽  
Wild Type ◽  
Cell Recruitment ◽  
Cell Dysfunction ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Zinc Binding Site ◽  
Secretory Phenotype

Our earlier studies demonstrated that cysteine414- (zinc-binding site of mCRY1-) alanine mutant mCRY1 transgenic mice (Tg mice) exhibit diabetes characterized by the reduction of β-cell proliferation and by β-cell dysfunction, presumably caused by senescence-associated secretory phenotype- (SASP-) like characters of islets. Earlier studies also showed that atypical duct-like structures in the pancreas developed age-dependently in Tg mice. Numerous reports have described that karyopherin alpha 2 (KPNA2) is highly expressed in cancers of different kinds. However, details of the expression of KPNA2 in pancreatic ductal atypia and in normal pancreatic tissues remain unclear. To assess the feature of the expression of KPNA2 in the development of the ductal atypia and islet architectures, we scrutinized the pancreas of Tg mice histopathologically. Results showed that considerable expression of KPNA2 was observed in pancreatic β-cells, suggesting its importance in maintaining the functions of β-cells. In mature stages, the level of KPNA2 expression was lower in islets of Tg mice than in wild-type controls. At 4 weeks, the expression levels of KPNA2 in islets of Tg mice were the same as those in wild-type controls. These results suggest that the reduction of KPNA2 might contribute to β-cell dysfunction in mature Tg mice. Additionally, the formation of mucin-producing intra-islet ducts, islet fibrosis, and massive T cell recruitment to the islet occurred in aged Tg mice. In exocrine areas, primary pancreatic intraepithelial neoplasias (PanINs) with mucinous pancreatic duct glands (PDGs) emerged in aged Tg mice. High expression of KPNA2 was observed in the ductal atypia. By contrast, KPNA2 expression in normal ducts was quite low. Thus, upregulation of KPNA2 seemed to be correlated with progression of the degree of atypia in pancreatic ductal cells. The SASP-like microenvironment inside islets might play stimulatory roles in the formation of ductal metaplasia inside islets and in islet fibrosis in Tg mice.

Effects of cholecystokinin on cytosolic calcium in pancreatic duct segments and ductal cells

1993 ◽  
Vol 264 (6) ◽  
pp. G1177-G1183 ◽  
Author(s):  
J. P. Smith ◽  
R. V. Yelamarty ◽  
S. T. Kramer ◽  
J. Y. Cheung
Keyword(s):  
Receptor Antagonist ◽  
Pancreatic Duct ◽  
Single Cells ◽  
Cytosolic Calcium ◽  
Extracellular Calcium ◽  
Pancreatic Ducts ◽  
Cck Receptors ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Water Secretion

Although the gastrointestinal peptide cholecystokinin (CCK) has been shown to increase bicarbonate and water secretion and potentiate the effects of secretin on pancreatic ducts, CCK receptors have not been identified on pancreatic ductal epithelium. The effects of CCK octapeptide (CCK-8) on cytosolic calcium were evaluated on microscopically dissected rat pancreatic duct segments and single rat duct cells from the established ARIP cell line. By use of fluorescence microscopy in fura-2-loaded duct segments or single cells, intracellular calcium concentration was measured in response to CCK-8 (4 x 10(-12)-4 x 10(-8) M). CCK-8 significantly increased cytosolic calcium up to 50-fold over baseline. The greatest increase occurred with the highest concentration of CCK-8 (4 x 10(-8) M). Oscillations were observed in experiments performed in buffer containing 0.68 mM physiological calcium. In another series of experiments performed in the presence of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to deplete extracellular calcium, CCK-8 treatment still resulted in significant increases in cytosolic calcium; however, oscillations were abolished. Since cytosolic calcium increased in the absence of extracellular calcium, the initial calcium rise most likely was from cytosolic stores. Our findings of CCK-8-stimulated increases in cytosolic calcium in microdissected pancreatic ducts suggest the presence of CCK receptors, a characteristic that was not lost in cultured pancreatic ductal cells. In addition, in ARIP cells, the CCK-8-induced increase in cytosolic calcium was abolished by pretreatment with the selective CCK-B receptor antagonist L-365,260 but not by the CCK-A receptor antagonist L-364,718.

scholarly journals Mutations in Residues Involved in Zinc Binding in the Catalytic Site of Escherichia coli Threonyl-tRNA Synthetase Confer a Dominant Lethal Phenotype

2007 ◽  
Vol 189 (19) ◽  
pp. 6839-6848 ◽  
Author(s):  
Joël Caillet ◽  
Monique Graffe ◽  
Flore Eyermann ◽  
Pascale Romby ◽  
Mathias Springer
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Dominant Negative ◽  
Zinc Binding ◽  
Regulatory Function ◽  
Dominant Negative Effect ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Zinc Binding Site ◽  
Negative Effect

ABSTRACT Escherichia coli threonyl-tRNA synthetase is a homodimeric protein that acts as both an enzyme and a regulator of gene expression: the protein aminoacylates tRNAThr isoacceptors and binds to its own mRNA, inhibiting its translation. The enzyme contains a zinc atom in its active site, which is essential for the recognition of threonine. Mutations in any of the three amino acids forming the zinc-binding site inactivate the enzyme and have a dominant negative effect on growth if the corresponding genes are placed on a multicopy plasmid. We show here that this particular property is not due to the formation of inactive heterodimers, the titration of tRNAThr by an inactive enzyme, or its misaminoacylation but is, rather, due to the regulatory function of threonyl-tRNA synthetase. Overproduction of the inactive enzyme represses the expression of the wild-type chromosomal copy of the gene to an extent incompatible with bacterial growth.

scholarly journals Unique Aspects of Cryptochrome in Chronobiology and Metabolism, Pancreaticβ-Cell Dysfunction, and Regeneration: Research into Cysteine414-Alanine Mutant CRY1

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Satoshi Okano
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Cell Loss ◽  
Zinc Binding ◽  
Beta Cell Dysfunction ◽  
Molecular Processes ◽  
Cell Dysfunction ◽  
New Information ◽  
Regulation Of Metabolism ◽  
Zinc Binding Site ◽  
Age Dependent

Cryptochrome proteins (CRYs), which can bind noncovalently to cofactor (chromophore) flavin adenine dinucleotide (FAD), occur widely among organisms. CRYs play indispensable roles in the generation of circadian rhythm in mammals. Transgenic mice (Tg mice), ubiquitously expressing mouse CRY1 having a mutation in which cysteine414 (the zinc-binding site of CRY1) being replaced with alanine, display unique phenotypes in their circadian rhythms. Moreover, male Tg mice exhibit symptoms of diabetes characterized by beta-cell dysfunction, resembling human maturity onset diabetes of the young (MODY). The lowered proliferation ofβ-cells is a primary cause of age-dependentβ-cell loss. Furthermore, unusually enlarged duct-like structures developed prominently in the Tg mice pancreases. The duct-like structures contained insulin-positive cells, suggesting neogenesis ofβ-cells in the Tg mice. This review, based mainly on the author’s investigation of the unique features of Tg mice, presents reported results and recent findings related to molecular processes associated with mammalian cryptochromes, especially their involvement in the regulation of metabolism. New information is described with emphasis on the aspects of islet architecture, pancreaticβ-cell dysfunction, and regeneration.

scholarly journals Intraislet Pancreatic Ducts Can Give Rise to Insulin-Positive Cells

Endocrinology ◽  
2016 ◽  
Vol 157 (1) ◽  
pp. 166-175 ◽  
Author(s):  
Yousef El-Gohary ◽  
John Wiersch ◽  
Sidhartha Tulachan ◽  
Xiangwei Xiao ◽  
Ping Guo ◽  
...  
Keyword(s):  
Lineage Tracing ◽  
Mutant Mice ◽  
Pancreatic Ducts ◽  
Diabetes Research ◽  
Wild Type ◽  
Β Cells ◽  
Partial Pancreatectomy ◽  
Ductal Cells ◽  
Adult Mice ◽  
Pancreatic Ductal Cells

Abstract A key question in diabetes research is whether new β-cells can be derived from endogenous, nonendocrine cells. The potential for pancreatic ductal cells to convert into β-cells is a highly debated issue. To date, it remains unclear what anatomical process would result in duct-derived cells coming to exist within preexisting islets. We used a whole-mount technique to directly visualize the pancreatic ductal network in young wild-type mice, young humans, and wild-type and transgenic mice after partial pancreatectomy. Pancreatic ductal networks, originating from the main ductal tree, were found to reside deep within islets in young mice and humans but not in mature mice or humans. These networks were also not present in normal adult mice after partial pancreatectomy, but TGF-β receptor mutant mice demonstrated formation of these intraislet duct structures after partial pancreatectomy. Genetic and viral lineage tracings were used to determine whether endocrine cells were derived from pancreatic ducts. Lineage tracing confirmed that pancreatic ductal cells can typically convert into new β-cells in normal young developing mice as well as in adult TGF-β signaling mutant mice after partial pancreatectomy. Here the direct visual evidence of ducts growing into islets, along with lineage tracing, not only represents strong evidence for duct cells giving rise to β-cells in the postnatal pancreas but also importantly implicates TGF-β signaling in this process.

pH regulatory mechanisms in rat pancreatic ductal cells

1988 ◽  
Vol 254 (6) ◽  
pp. G925-G930 ◽  
Author(s):  
E. L. Stuenkel ◽  
T. E. Machen ◽  
J. A. Williams
Keyword(s):  
Pancreatic Duct ◽  
Exchange Mechanism ◽  
Disulfonic Acid ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells ◽  
Duct Cells ◽  
Pancreatic Duct Cells ◽  
Stilbene Derivatives ◽  
Disulfonic Stilbene ◽  
Intracellular Alkalinization

The mechanisms underlying regulation of intracellular pH (pHi) by rat pancreatic duct cells were studied by use of the pH-sensitive, fluorescent, cytoplasmically trapped dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Cells exhibited a mean pHi of 7.18 +/- 0.14 in bicarbonate-buffered medium, as calculated from the BCECF fluorescence ratio. Removal of extracellular Na (Nao) caused an intracellular acidification that was rapidly reversed by Na replacement and occurred independently of Clo. Amiloride (10(-3) M) reversibly blocked Na-dependent recovery after Na-free-induced acidification. These results demonstrate the presence of a Na+-H+ exchange mechanism in pancreatic duct cells. Replacement of Clo with gluconate caused an intracellular alkalinization that was reversed by replacement of Cl. Application of the disulfonic stilbene derivatives, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and dihydro-DIDS (2 X 10(-4) M), resulted in block of both Cl-dependent recovery from Na-gluconate and the onset of alkalinization of transition from NaCl to Na-gluconate. Chloride-dependent alteration of pHi occurred independently of Nao. These results demonstrate the presence of an anion exchange mechanism consistent with Cl--HCO3- exchange. Thus pancreatic duct cells contain both Na+-H+ and Cl--HCO3- exchangers.

Isolation and culture of bovine pancreatic duct epithelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. G1328-G1337 ◽  
Author(s):  
C. U. Cotton ◽  
L. al-Nakkash
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Pancreatic Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Intracellular Camp ◽  
Bathing Solution ◽  
Anion Secretion ◽  
Ductal Cells ◽  
Pancreatic Ductal Cells

We describe a method to isolate and culture epithelial cells from the main duct of the bovine pancreas. In primary cultures, secretin caused a dose-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and stimulated electrogenic transepithelial ion transport. Elevation of intracellular cAMP increased the rate coefficient for 36Cl- efflux from 0.14 +/- 0.03 to 0.47 +/- 0.12 min-1, and plasma membrane conductance, measured by the whole cell patchclamp technique, was increased from 0.7 +/- 0.1 to 6.9 +/- 0.8 nS. The cAMP-activated anion currents had properties similar to those mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Cells grown on permeable supports formed confluent monolayers with high transepithelial electrical resistance (1.004 +/- 96 omega. cm2) and generated a lumen negative transepithelial voltage difference (-2.5 +/- 0.6 mV). The short-circuit current (Isc) was increased by forskolin or secretin and was inhibited 87 +/- 4% by addition of ouabain (100 microM) to the basolateral bathing solution. Replacement of bathing solution Cl- by cyclamate reduced the forskolin-induced steady-state increase in Isc from 5.3 +/- 0.5 to 0.2 +/- 0.2 microA/cm2, suggesting that the stimulated current is due to anion secretion. The results of these studies demonstrate that large numbers of pancreatic ductal cells can be isolated and grown in primary cell culture. The monolayers express differentiated functions and will be useful for studies of acute and chronic regulation of ion transport in pancreatic duct epithelial cells.

scholarly journals Stability of Cucumber Necrosis Virus at the Quasi-6-Fold Axis Affects Zoospore Transmission

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Michael B. Sherman ◽  
Kishore Kakani ◽  
D'Ann Rochon ◽  
Wen Jiang ◽  
Neil R. Voss ◽  
...  
Keyword(s):  
Plant Viruses ◽  
Zinc Binding ◽  
Wild Type ◽  
Binding Region ◽  
Necrosis Virus ◽  
Content Type ◽  
Link Type ◽  
Olpidium Bornovanus ◽  
Zinc Binding Site ◽  
Cucumber Necrosis Virus

ABSTRACT Cucumber necrosis virus (CNV) is a member of the genus Tombusvirus and has a monopartite positive-sense RNA genome. CNV is transmitted in nature via zoospores of the fungus Olpidium bornovanus. As with other members of the Tombusvirus genus, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507–517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008 ). P73 lies immediately adjacent to a putative zinc binding site (M. Li et al., J Virol 87:12166–12175, 2013, https://doi.org/10.1128/JVI.01965-13 ) that is formed by three icosahedrally related His residues in the N termini of the C subunit at the quasi-6-fold axes. To better understand how this buried residue might affect vector transmission, we determined the cryo-electron microscopy structure of wild-type CNV in the native and swollen state and of the transmission-defective mutant, P73G, under native conditions. With the wild-type CNV, the swollen structure demonstrated the expected expansion of the capsid. However, the zinc binding region at the quasi-6-fold at the β-annulus axes remained intact. By comparison, the zinc binding region of the P73G mutant, even under native conditions, was markedly disordered, suggesting that the β-annulus had been disrupted and that this could destabilize the capsid. This was confirmed with pH and urea denaturation experiments in conjunction with electron microscopy analysis. We suggest that the P73G mutation affects the zinc binding and/or the β-annulus, making it more fragile under neutral/basic pH conditions. This, in turn, may affect zoospore transmission. IMPORTANCE Cucumber necrosis virus (CNV), a member of the genus Tombusvirus, is transmitted in nature via zoospores of the fungus Olpidium bornovanus. While a number of plant viruses are transmitted via insect vectors, little is known at the molecular level as to how the viruses are recognized and transmitted. As with many spherical plant viruses, the CNV capsid swells when exposed to alkaline pH and EDTA. We previously demonstrated that a P73G mutation that lies inside the capsid immediately adjacent to a putative zinc binding site (Li et al., J Virol 87:12166–12175, 2013, https://doi.org/10.1128/JVI.01965-13 ) blocks the virus from zoospore transmission while not significantly affecting replication in plants (K. Kakani, R. Reade, and D. Rochon, J Mol Biol 338:507–517, 2004, https://doi.org/10.1016/j.jmb.2004.03.008 ). Here, we show that the P73G mutant is less stable than the wild type, and this appears to be correlated with destabilization of the β-annulus at the icosahedral 3-fold axes. Therefore, the β-annulus appears not to be essential for particle assembly but is necessary for interactions with the transmission vector.

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