scholarly journals A Protein Kinase A-Dependent Mechanism by Which Rotavirus Affects the Distribution and mRNA Level of the Functional Tight Junction-Associated Protein, Occludin, in Human Differentiated Intestinal Caco-2 Cells

2007 ◽  
Vol 81 (16) ◽  
pp. 8579-8586 ◽  
Author(s):  
Isabelle Beau ◽  
Jacqueline Cotte-Laffitte ◽  
Raymonde Amsellem ◽  
Alain L. Servin
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Laser Scanning ◽  
Time Course ◽  
Mrna Level ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Infected Cells ◽  
Triton X ◽  
Kinase A

ABSTRACT We found that at the tight junctions (TJs) of Caco-2 cell monolayers, rhesus monkey rotavirus (RRV) infection induced the disappearance of occludin. Confocal laser scanning microscopy showed the disappearance of occludin from the cell-cell boundaries without modifying the expression of the other TJ-associated proteins, ZO-1 and ZO-3. Western immunoblot analysis of RRV-infected cells showed a significant fall in the levels of the nonphosphorylated form of occludin in both Triton X-100-insoluble and Triton X-100-soluble fractions, without any change in the levels of the phosphorylated form of occludin. Quantitative reverse transcription-PCRs revealed that the level of transcription of the gene that encodes occludin was significantly reduced in RRV-infected cells. Treatment of RRV-infected cells with Rp-cyclic AMP and protein kinase A inhibitors H89 and KT5720 during the time course of the infection restored the distribution of occludin and a normal level of transcription of the gene that encodes occludin.

scholarly journals The Role of Uncoupling Protein 1 in the Metabolism and Adiposity of RIIβ-Protein Kinase A-Deficient Mice

2004 ◽  
Vol 18 (9) ◽  
pp. 2302-2311 ◽  
Author(s):  
Michael A. Nolan ◽  
Maria A. Sikorski ◽  
G. Stanley McKnight
Keyword(s):  
Adipose Tissue ◽  
Oxygen Consumption ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Uncoupling Protein ◽  
Mrna Level ◽  
Regulatory Subunit ◽  
Uncoupling Protein 1 ◽  
Brown Adipose ◽  
Kinase A

Abstract Mice lacking the RIIβ regulatory subunit of protein kinase A exhibit a 50% reduction in white adipose tissue stores compared with wild-type littermates and are resistant to diet-induced obesity. RIIβ−/− mice also have an increase in resting oxygen consumption along with a 4-fold increase in the brown adipose-specific mitochondrial uncoupling protein 1 (UCP1). In this study, we examined the basis for UCP1 induction and tested the hypothesis that the induced levels of UCP1 in RIIβ null mice are essential for the lean phenotype. The induction of UCP1 occurred at the protein but not the mRNA level and correlated with an increase in mitochondria in brown adipose tissue. Mice lacking both RIIβ and UCP1 (RIIβ−/−/Ucp1−/−) were created, and the key parameters of metabolism and body composition were studied. We discovered that RIIβ−/− mice exhibit nocturnal hyperactivity in addition to the increased oxygen consumption at rest. Disruption of UCP1 in RIIβ−/− mice reduced basal oxygen consumption but did not prevent the nocturnal hyperactivity. The double knockout animals also retained the lean phenotype of the RIIβ null mice, demonstrating that induction of UCP1 and increased resting oxygen consumption is not the cause of leanness in the RIIβ mutant mice.

The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 379-388 ◽  
Author(s):  
M.J. Carette ◽  
M.W. Ferguson
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Palatal Fusion ◽  
Morphological Criteria ◽  
Medial Edge ◽  
Mesenchymal Transformation

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.

Tyrosine phosphorylation following alterations in arteriolar intraluminal pressure and wall tension

2001 ◽  
Vol 281 (3) ◽  
pp. H1047-H1056 ◽  
Author(s):  
Timothy V. Murphy ◽  
Brian E. Spurrell ◽  
Michael A. Hill
Keyword(s):  
Tyrosine Phosphorylation ◽  
Fluorescence Intensity ◽  
Laser Scanning ◽  
Time Course ◽  
Transmural Pressure ◽  
Laser Scanning Microscopy ◽  
Intraluminal Pressure ◽  
Confocal Laser ◽  
Wall Tension ◽  
Myogenic Activity

Arterioles respond to increased transmural pressure with myogenic constriction. The present study investigated the role of tyrosine phosphorylation in myogenic activity. Cannulated segments of a rat cremaster arteriole were fixed under pressure, followed by incubation with fluorescein isothiocyanate (FITC)-conjugated anti-phosphotyrosine. Smooth muscle cell fluorescence intensity was measured with the use of confocal laser-scanning microscopy. Anti-phosphotyrosine fluorescence intensity in muscle cells of arterioles maintained at 100 mmHg was reduced by the tyrosine kinase inhibitor tyrphostin A47 (30 μM) and increased by the tyrosine phosphatase inhibitor pervanadate (100 μM). In time-course experiments, anti-phosphotyrosine fluorescence increased slowly (over 5 min) after an acute increase in intraluminal pressure, and was dissociated from myogenic contraction (within 1 min). In contrast, angiotensin II (0.1 μM) caused rapid constriction and increased tyrosine phosphorylation. Anti-phosphotyrosine fluorescence was also pressure dependent (10–100 mmHg). Abolition of myogenic activity, either through removal of extracellular Ca2+, or exposure to verapamil (5 μM) or forskolin (0.1 μM) caused a further increase in anti-phosphotyrosine fluorescence. We conclude that transmural pressure and/or wall tension in arterioles causes increased tyrosine phosphorylation; however, this is not involved in the acute phase of myogenic constriction but may be involved in later responses, such as sustained myogenic tone or mechanisms possibly related to growth.

scholarly journals Chronic opioids regulate KATP channel subunit Kir6.2 and carbonic anhydrase I and II expression in rat adrenal chromaffin cells via HIF-2α and protein kinase A

2014 ◽  
Vol 307 (3) ◽  
pp. C266-C277 ◽  
Author(s):  
Shaima Salman ◽  
Alison C. Holloway ◽  
Colin A. Nurse
Keyword(s):  
Protein Kinase ◽  
Carbonic Anhydrase ◽  
Protein Kinase A ◽  
Chromaffin Cells ◽  
Time Course ◽  
Molecular Mechanisms ◽  
Hypoxia Inducible Factor ◽  
Neonatal Rat ◽  
Pcr Analysis ◽  
Kinase A

At birth, asphyxial stressors such as hypoxia and hypercapnia are important physiological stimuli for adrenal catecholamine release that is critical for the proper transition to extrauterine life. We recently showed that chronic opioids blunt chemosensitivity of neonatal rat adrenomedullary chromaffin cells (AMCs) to hypoxia and hypercapnia. This blunting was attributable to increased ATP-sensitive K+ (KATP) channel and decreased carbonic anhydrase (CA) I and II expression, respectively, and involved μ- and δ-opioid receptor signaling pathways. To address underlying molecular mechanisms, we first exposed an O2- and CO2-sensitive, immortalized rat chromaffin cell line (MAH cells) to combined μ {[d-Arg2,Ly4]dermorphin-(1–4)-amide}- and δ ([d-Pen2,5,P-Cl-Phe4]enkephalin)-opioid agonists (2 μM) for ∼7 days. Western blot and quantitative real-time PCR analysis revealed that chronic opioids increased KATP channel subunit Kir6.2 and decreased CAII expression; both effects were blocked by naloxone and were absent in hypoxia-inducible factor (HIF)-2α-deficient MAH cells. Chronic opioids also stimulated HIF-2α accumulation along a time course similar to Kir6.2. Chromatin immunoprecipitation assays on opioid-treated cells revealed the binding of HIF-2α to a hypoxia response element in the promoter region of the Kir6.2 gene. The opioid-induced regulation of Kir6.2 and CAII was dependent on protein kinase A, but not protein kinase C or calmodulin kinase, activity. Interestingly, a similar pattern of HIF-2α, Kir6.2, and CAII regulation (including downregulation of CAI) was replicated in chromaffin tissue obtained from rat pups born to dams exposed to morphine throughout gestation. Collectively, these data reveal novel mechanisms by which chronic opioids blunt asphyxial chemosensitivity in AMCs, thereby contributing to abnormal arousal responses in the offspring of opiate-addicted mothers.

scholarly journals Effect of arsenite on swimming motility delays surface colonization in Herminiimonas arsenicoxydans

Microbiology ◽  
2010 ◽  
Vol 156 (8) ◽  
pp. 2336-2342 ◽  
Author(s):  
M. Marchal ◽  
R. Briandet ◽  
S. Koechler ◽  
B. Kammerer ◽  
P. N. Bertin
Keyword(s):  
Laser Scanning ◽  
Time Course ◽  
Wild Type Strain ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wild Type ◽  
Arsenite Oxidase ◽  
Swimming Motility ◽  
In The Wild ◽  
Biofilm Development

Herminiimonas arsenicoxydans is a Gram-negative bacterium able to detoxify arsenic-contaminated environments by oxidizing arsenite [As(III)] to arsenate [As(V)] and by scavenging arsenic ions in an extracellular matrix. Its motility and colonization behaviour have been previously suggested to be influenced by arsenite. Using time-course confocal laser scanning microscopy, we investigated its biofilm development in the absence and presence of arsenite. Arsenite was shown to delay biofilm initiation in the wild-type strain; this was partly explained by its toxicity, which caused an increased growth lag time. However, this delayed adhesion step in the presence of arsenite was not observed in either a swimming motility defective fliL mutant or an arsenite oxidase defective aoxB mutant; both strains displayed the wild-type surface properties and growth capacities. We propose that during the biofilm formation process arsenite acts on swimming motility as a result of the arsenite oxidase activity, preventing the switch between planktonic and sessile lifestyles. Our study therefore highlights the existence, under arsenite exposure, of a competition between swimming motility, resulting from arsenite oxidation, and biofilm initiation.

Transcript of protein kinase A knock-down modulates feeding behavior and neuropeptide Y gene expression in phenylpropanolamine-treated rats

2007 ◽  
Vol 31 (2) ◽  
pp. 306-314 ◽  
Author(s):  
Yih-Shou Hsieh ◽  
Shun-Fa Yang ◽  
Shu-Chen Chu ◽  
Dong-Yih Kuo
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Neuropeptide Y ◽  
Protein Kinase A ◽  
Mrna Level ◽  
Camp Response Element Binding ◽  
Antisense Oligodeoxynucleotide ◽  
Mrna Levels ◽  
Knock Down ◽  
Kinase A

Neuropeptide Y (NPY) is an appetite-controlling neuromodulator that contributes to the appetite-suppressing effect of phenylpropanolamine (PPA). Aims of this study were to investigate whether protein kinase A (PKA) signaling is involved in regulating NPY gene expression and PPA-induced anorexia. Rats were given daily with PPA for 5 days. Changes in daily food intake and hypothalamic NPY, PKA, cAMP response element binding protein (CREB), and pro-opiomelanocortin (POMC) gene expression were measured and compared. To further determine if PKA was involved, intracerebroventricular infusions of antisense oligodeoxynucleotide were performed at 60 min before daily PPA treatment in freely moving rats. Results showed that daily PKA, CREB, and POMC expression were increased following PPA treatment, which showed a closely reverse relationship with alterations of decreased feeding behaviors and NPY mRNA levels. Results also showed that PKA knock-down could block PPA-induced anorexia as well as restore NPY mRNA level, indicating the involvement of PKA signaling in the regulation of NPY gene expression. It is suggested that hypothalamic PKA signaling may participate in the central regulation of PPA-mediated appetite suppression via the modulation of hypothalamic NPY gene expression. The present findings reveal that manipulations at the molecular level of PKA or cAMP may allow the development of therapeutic agents to improve the undesirable properties of PPA or other amphetamine-like anorectic drugs.

scholarly journals Protein Kinase A-Dependent Derepression of the Human Prodynorphin Gene via Differential Binding to an Intragenic Silencer Element

1998 ◽  
Vol 18 (12) ◽  
pp. 6921-6929 ◽  
Author(s):  
Angel M. Carrión ◽  
Britt Mellström ◽  
Jose R. Naranjo
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Time Course ◽  
Neuroblastoma Cells ◽  
Site Directed Mutagenesis ◽  
Deletion Mapping ◽  
Human Neuroblastoma Cells ◽  
Basal Transcription ◽  
Human Neuroblastoma ◽  
Kinase A

ABSTRACT Induction of the prodynorphin gene has been implicated in medium and long-term adaptation during memory acquisition and pain. By 5′ deletion mapping and site-directed mutagenesis of the human prodynorphin promoter, we demonstrate that both basal transcription and protein kinase A (PKA)-induced transcription in NB69 and SK-N-MC human neuroblastoma cells are regulated by the GAGTCAAGG sequence centered at position +40 in the 5′ untranslated region of the gene (named the DRE, for downstream regulatory element). The DRE repressed basal transcription in an orientation-independent and cell-specific manner when placed downstream from the heterologous thymidine kinase promoter. Southwestern blotting and UV cross-linking experiments with nuclear extracts from human neuroblastoma cells or human brain revealed a protein complex of approximately 110 kDa that specifically bound to the DRE. Forskolin treatment reduced binding to the DRE, and the time course paralleled that for an increase in prodynorphin gene expression. Our results suggest that under basal conditions, expression of the prodynorphin gene is repressed by occupancy of the DRE site. Upon PKA stimulation, binding to the DRE is reduced and transcription increases. We propose a model for human prodynorphin activation through PKA-dependent derepression at the DRE site.

scholarly journals Adrenergic Regulation and Diurnal Rhythm of p38 Mitogen-Activated Protein Kinase Phosphorylation in the Rat Pineal Gland

Endocrinology ◽  
2004 ◽  
Vol 145 (11) ◽  
pp. 5194-5201 ◽  
Author(s):  
C. L. Chik ◽  
M. Mackova ◽  
D. Price ◽  
A. K. Ho
Keyword(s):  
Protein Kinase ◽  
Pineal Gland ◽  
Protein Kinase A ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Adrenergic Blocker ◽  
Rat Pineal Gland ◽  
Kinase A

Abstract In this study, we investigated adrenergic and photoneural regulation of p38MAPK phosphorylation in the rat pineal gland. Norepinephrine (NE), the endogenous neurotransmitter, dose-dependently increased the levels of phosphorylated MAPK kinase 3/6 (MKK3/6) and p38MAPK in rat pinealocytes. Time-course studies showed a gradual increase in MKK3/6 and p38MAPK phosphorylation that peaked between 1 and 2 h and persisted for 4 h post NE stimulation. In cells treated with NE for 2 and 4 h, the inclusion of prazosin or propranolol reduced NE-induced MKK3/6 and p38MAPK phosphorylation, indicating involvement of both α- and β-adrenergic receptors for the sustained response. Whereas treatment with dibutyryl cAMP or ionomycin mimicked the NE-induced MKK3/6 and p38MAPK phosphorylation, neither dibutyryl cGMP nor 4β-phorbol 12-myristate 13-acetate had an effect. The NE-induced increase in MKK3/6 and p38MAPK phosphorylation was blocked by KT5720 (a protein kinase A inhibitor) and KN93 (a Ca2+/calmodulin-dependent kinase inhibitor), but not by KT5823 (a protein kinase G inhibitor) or calphostin C (a protein kinase C inhibitor). In animals housed under a lighting regimen with 12 h of light, MKK3/6 and p38MAPK phosphorylation increased in the rat pineal gland at zeitgeber time 18. The nocturnal increase in p38MAPK phosphorylation was blocked by exposing the animal to constant light and reduced by treatment with propranolol, a β-adrenergic blocker. Together, our results indicate that activation of p38MAPK is under photoneural control in the rat pineal gland and that protein kinase A and intracellular Ca2+ signaling pathways are involved in NE regulation of p38MAPK.

Phosphorylated baculovirus p10 is a heat-stable microtubule-associated protein associated with process formation in Sf9 cells

1992 ◽  
Vol 102 (4) ◽  
pp. 739-752
Author(s):  
S. Cheley ◽  
K.S. Kosik ◽  
P. Paskevich ◽  
S. Bakalis ◽  
H. Bayley
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Recombinant Baculovirus ◽  
Catalytic Subunit ◽  
Sf9 Cells ◽  
Wild Type ◽  
Heat Stable ◽  
Process Formation ◽  
Infected Cells ◽  
Kinase A

Insect ovarian Sf9 cells extend processes with complex morphologies when infected with a recombinant baculovirus encoding the catalytic subunit of protein kinase A. Within the shafts of the processes are abundant microtubules, which, in contrast to those in Sf9 cells expressing the microtubule-associated protein tau, are generally not organized into parallel bundles. During infection the late viral polypeptide p10 becomes phosphorylated by the protein kinase A catalytic subunit at its penultimate residue, Ser92. The expression or phosphorylation of other major host cell or viral polypeptides does not change, compared with polypeptides from a wild-type viral infection. Once phosphorylated, p10 associates with microtubules in the infected cells and may thereby play a role in process formation.

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