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Salivary Gland Protein Expression after Bion-M1 and Space Shuttle STS-135 Missions

Maija Mednieks

Maija Mednieks

Department of Oral Health and Diagnostic SciencesFlorida, United States
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Mednieks, Maija
, 
Aditi Khatri

Aditi Khatri

Department of Oral Health and Diagnostic SciencesFlorida, United States
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Khatri, Aditi
 und 
Arthur R. Hand

Arthur R. Hand

Department of Craniofacial Sciences, University of Connecticut School of Dental MedicineFarmington, CT, United States
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Hand, Arthur R.
  
01. Juli 2015
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Artikel-Kategorie: Research Article
Online veröffentlicht: 01. Juli 2015
Seitenbereich: 2 - 19
DOI: https://doi.org/10.2478/gsr-2015-0001
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© 2015 Maija Mednieks et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Figure 1.

Cyclic AMP-PKA signaling pathways of regulated exocytosis in cell compartments. Schematic representation of cell surface beta-adrenergic receptor stimulation, signal transduction, cyclic AMP second messenger cytoplasmic and nuclear response. βAR, beta-adrenergic receptor; Gs, heterotrimeric G protein; AC, adenylate cyclase; PDE, phosphodiesterase; AKAP, A-kinase anchor protein; RII, type II PKA regulatory subunit; C, PKA catalytic subunit; ATF1, activating transcription factor 1; CREB, cyclic AMP response element binding protein; CREM, cyclic AMP responsive element modulator. The large blue sphere represents a secretory granule along a path to fuse with the cell membrane and to empty its contents of secretory proteins into the lumen.
Cyclic AMP-PKA signaling pathways of regulated exocytosis in cell compartments. Schematic representation of cell surface beta-adrenergic receptor stimulation, signal transduction, cyclic AMP second messenger cytoplasmic and nuclear response. βAR, beta-adrenergic receptor; Gs, heterotrimeric G protein; AC, adenylate cyclase; PDE, phosphodiesterase; AKAP, A-kinase anchor protein; RII, type II PKA regulatory subunit; C, PKA catalytic subunit; ATF1, activating transcription factor 1; CREB, cyclic AMP response element binding protein; CREM, cyclic AMP responsive element modulator. The large blue sphere represents a secretory granule along a path to fuse with the cell membrane and to empty its contents of secretory proteins into the lumen.

Figure 2.

Ultrastructure of parotid glands of flight mice. Panel A, STS-135: Acinar cells have basally located nuclei [N (binucleate cells are common)], a prominent Golgi complex (G), and abundant apically located secretory granules (SG). Intercellular canaliculus (IC). Scale bar = 2 μm. Panel B, STS-135: Intercalated duct. The duct cells have apical vacuoles (arrows) containing material endocytosed from the lumen (L). Scale bar = 2 μm. The inset shows a similar vacuole in a striated duct cell immunogold labeled for PSP, an acinar secretory protein. Scale bar = 0.5 μm. Panel C, Bion-M1: A recently formed autophagic vacuole (AV1) containing secretory proteins labeled for PSP is present in an acinar cell. An older autophagic vacuole (AV2) contains remnants of secretory granules and other organelles, but shows little labeling. Secretory granule (SG); nucleus (N). Scale bar = 1 μm. Panel D, Bion-M1: Apoptotic acinar cells (Apop) phagocytosed by a macrophage (arrowheads). Myoepithelial cell (MEC). Scale bar = 2 μm.
Ultrastructure of parotid glands of flight mice. Panel A, STS-135: Acinar cells have basally located nuclei [N (binucleate cells are common)], a prominent Golgi complex (G), and abundant apically located secretory granules (SG). Intercellular canaliculus (IC). Scale bar = 2 μm. Panel B, STS-135: Intercalated duct. The duct cells have apical vacuoles (arrows) containing material endocytosed from the lumen (L). Scale bar = 2 μm. The inset shows a similar vacuole in a striated duct cell immunogold labeled for PSP, an acinar secretory protein. Scale bar = 0.5 μm. Panel C, Bion-M1: A recently formed autophagic vacuole (AV1) containing secretory proteins labeled for PSP is present in an acinar cell. An older autophagic vacuole (AV2) contains remnants of secretory granules and other organelles, but shows little labeling. Secretory granule (SG); nucleus (N). Scale bar = 1 μm. Panel D, Bion-M1: Apoptotic acinar cells (Apop) phagocytosed by a macrophage (arrowheads). Myoepithelial cell (MEC). Scale bar = 2 μm.

Figure 3.

Immunogold labeling of parotid acinar cell secretory granules. Panel A, STS-135: Flight, PRPs. Panel B, STS-135: AEM ground control, PRPs. Panel C, Bion-M1: Flight, amylase. Panel D, Bion-M1: Habitat ground control, amylase. Intercellular canaliculus (IC); mitochondrion (M). Scale bar = 0.5 μm.
Immunogold labeling of parotid acinar cell secretory granules. Panel A, STS-135: Flight, PRPs. Panel B, STS-135: AEM ground control, PRPs. Panel C, Bion-M1: Flight, amylase. Panel D, Bion-M1: Habitat ground control, amylase. Intercellular canaliculus (IC); mitochondrion (M). Scale bar = 0.5 μm.

Figure 4.

Morphometric analysis of immunogold labeling of parotid secretory proteins. Percent change in STS-135 (light bars) and Bion-M1 (dark bars) flight mice from habitat controls.
Morphometric analysis of immunogold labeling of parotid secretory proteins. Percent change in STS-135 (light bars) and Bion-M1 (dark bars) flight mice from habitat controls.

Figure 5.

Electrophoresis, Western blotting, and densitometry of parotid proteins. Panel A, Bion-M1 control and flight parotid soluble proteins. The lanes are molecular size marker (M), flight (F), and vivarium control (C). The two bands on the Western blot (WB) are at the mobility of RII and an RII fragment (Rfr), respectively. Panel B, Densitometry of the banding pattern profiles. Vivarium control (light gray curve) superimposed with the flight (darker curve) protein electrophoretic pattern. The ordinate shows integrated density (ID) values and the abscissa shows the calculated molecular size in kilo Dalton (kD) units.
Electrophoresis, Western blotting, and densitometry of parotid proteins. Panel A, Bion-M1 control and flight parotid soluble proteins. The lanes are molecular size marker (M), flight (F), and vivarium control (C). The two bands on the Western blot (WB) are at the mobility of RII and an RII fragment (Rfr), respectively. Panel B, Densitometry of the banding pattern profiles. Vivarium control (light gray curve) superimposed with the flight (darker curve) protein electrophoretic pattern. The ordinate shows integrated density (ID) values and the abscissa shows the calculated molecular size in kilo Dalton (kD) units.

Figure 6.

Densitometric analysis of polyacrylamide gel electrophoretic protein separation and anti-RII and anti-α-amylase reactivity in STS-135 mice. Panel A: Top panel, flight; middle panel, vivarium; and bottom panel, habitat parotid gland samples. The lighter curve represents the protein profile; the shaded area is the reactivity to anti-RII antibody. The ordinate axes are integrated density values of the proteins on the left hand axis, and the integrated density values for RII and the reactivity of RII fragment (Rfr) on the right hand axis. Panel B shows parotid α-amylase Western blotting. The ordinate shows integrated density, and the error bars show ± 5% error. F, flight; C1, vivarium control; C2, habitat control.
Densitometric analysis of polyacrylamide gel electrophoretic protein separation and anti-RII and anti-α-amylase reactivity in STS-135 mice. Panel A: Top panel, flight; middle panel, vivarium; and bottom panel, habitat parotid gland samples. The lighter curve represents the protein profile; the shaded area is the reactivity to anti-RII antibody. The ordinate axes are integrated density values of the proteins on the left hand axis, and the integrated density values for RII and the reactivity of RII fragment (Rfr) on the right hand axis. Panel B shows parotid α-amylase Western blotting. The ordinate shows integrated density, and the error bars show ± 5% error. F, flight; C1, vivarium control; C2, habitat control.

Figure 7.

Microarray analyses of selected parotid genes. The ordinate shows the log2 values of the ratio of the means of the normalized signal intensities determined for each gene for flight vs. habitat control. Prkar2a, protein kinase, cAMP dependent regulatory, type II alpha; Akap13, A-kinase anchor protein 13; Pde4a, phosphodiesterase 4a, cAMP specific; Adrb2, adrenergic receptor, beta 2; Adcy3, adenylate cyclase 3; Amy1, amylase 1, salivary.
Microarray analyses of selected parotid genes. The ordinate shows the log2 values of the ratio of the means of the normalized signal intensities determined for each gene for flight vs. habitat control. Prkar2a, protein kinase, cAMP dependent regulatory, type II alpha; Akap13, A-kinase anchor protein 13; Pde4a, phosphodiesterase 4a, cAMP specific; Adrb2, adrenergic receptor, beta 2; Adcy3, adenylate cyclase 3; Amy1, amylase 1, salivary.

Ratios of RII to Total Protein of STS-135 and Bion-M1 PAGE_

F C1 C2
STS-135 PAGE 70.8 69.28 69.99
RII 5.16 41.43 24.24
Ratio 0.073 0.60 0.35
Bion-M1 PAGE 153.6 129.0 ---
RII 21.7 16.26 ---
Ratio 0.14 0.13 ---

Quantitative Immunogold Labeling of STS-135 and Bion-M1 Salivary Glands (Gold Particles/µm 2 ± SEM)_

Bion-M1Parotid Flight Habitat
Amylase 15.3 ± 1.11∞ 10.3 ± 0.69
RII 10.1 ± 0.47 11.3 ± 0.49
PSP 37.4 ± 1.17§ 30.6 ± 1.76
PRP 22.5 ± 1.61 24.8 ± 2.08
DCPP 23.1 ± 0.95 25.2 ± 1.27

Microarray Analyses of Secretory Protein Gene Expression_

STS-135 Bion-M1
Gene a Log2 F/Hb Fold Change F/H Log2 F/H Fold Change F/H
Amy1 0.030 1.02 0.169* 1.124
Bpifa2 0.241§ 1.18 0.170* 1.13
Lpo 0.070 1.05 0.160* 1.18
Lyz1 0.317 1.25 0.898∞ 1.86
Pip 0.228* 1.17 0.169* 1.13
Prpmp5 -0.161§ 0.894 -0.861∞ 0.550

Body Weights of STS-135 and Bion-M1 Mice_

Group Initial Body Wt. (g) ± SEM (n) Final Body Wt. (g) ± SEM (n)
STS-135
    Flight 20.7 ± 0.40 (7) 18.4 ± 0.52 (7)
    AEM Ground Control 20.7 ± 0.31 (15) 19.4 ± 0.33 (15)
    Vivarium Ground Control --- 19.7 ± 0.34 (15)
Bion-M1
    Flight 26.8 ± 0.55 (6) 29.4 ± 1.73 (6)
    Synchronous Vivarium Ground Control 29.2 ± 0.44 (8) 28.7 ± 0.30 (8)
    Asynchronous Habitat Ground Control 27.3 ± 1.15 (7) 29.7 ± 0.83 (7)
    Asynchronous Vivarium Ground Control 26.7 ± 0.78 (7) 28.9 ± 1.03 (7)
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