*2.2.2 Electrophoresis and Western blotting*

Polyacrylamide gel electrophoresis (PAGE) is the separation of proteins based on mobility on a gel matrix, subjected to a charge differential between two sealed chambers, of a body fluid or tissue extract. This technique is frequently used to determine the distribution or banding pattern of protein groups in tissues under different conditions. Quantification of the intensity of the bands and their relative mobility by PAGE is carried out by densitometric analysis. Saliva is a convenient

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*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

peaks, but not the basic pattern.

**Figure 7.**

stability of the tissue or of sample preparation.

in the flight and in the habitat controls.

body fluid for measuring a variety of physiological conditions or responses on the basis of variations in protein content or distribution. For example, after testing more than 50 samples, PAGE separation of salivary proteins has shown that each individual pattern is unique. Namely, no superimposable patterns were found, therefore constituting essentially a salivary protein pattern fingerprint. Shown at the left in **Figure 7**, in duplicate, are the patterns of three (A, B, C) randomly selected individuals with the corresponding densitometric profiles at the right. **Figure 7** also shows how normal chewing changes the amplitude of several protein

*Electrophoresis and densitometry of saliva from three randomly selected individuals (A, B, and C) from a pool of approximately 50 test subjects. The left ordinate on the gel pattern image shows molecular based migration rates of a mixture of proteins of known size. At the right of the polyacrylamide gel electrophoresis (PAGE) banding patterns are the corresponding densitometric measurements. The top row (−) prior to, the bottom row (+) after, chewing. The asterisks show the migration distance of RII that can be identified by Western blotting.*

The PAGE protein banding patterns of the STS mission samples showed a decrease of some of the higher molecular weight bands while the faster moving smaller bands increased when these were compared to vivarium controls. The changes were not consistent between missions and may in part be due to lack of

Western blotting is a technique using antibodies generated to a specific protein for its identification among numerous others in the banding pattern. Proteins are transferred onto nitrocellulose membranes and stained with a washable dye to show the band distribution, then quantified by densitometry. The dye is removed and the membrane probed for reactivity with a specific antibody. These techniques were employed to test salivary gland extracts to measure protein patterns of flight animal

samples when compared with those of ground control animals (**Figure 8**).

The results show that the RII peaks in the STS-135 flight samples (**Figure 8**) were significantly smaller than the corresponding band amplitude in either vivarium or habitat controls, middle and bottom profiles, respectively. Western blotting experiments to determine the expression of α-amylase show a significant decrease

These results indicate that on the shorter, STS-135 flight the expression of RII was decreased, while in the longer Bion-M1 flight (**Figure 9**) an apparent stabilization or adjustment to the microgravity environment had occurred and the RII levels were not different, perhaps even increased from those of either of the controls. Both Bion-M1 and STS-135 flight and both control samples show a significant, but opposite change of a faster moving band that may represent a small protein or peptide. The findings regarding RII, generally thought to be a stress associated protein [35, 36] responding to the conditions of spaceflight [7], varied by methods of analysis but also between flights within a particular test measure from morphometric analyses of immunogold labeling experiments. Bion-M1 Western blotting results showed no difference between RII in Bion-M1 flight and vivarium control

**Figure 7.**

*Beyond LEO - Human Health Issues for Deep Space Exploration*

The expression of SMG secretory proteins was essentially unchanged in mice from the 13–15-day space shuttle flights. However, mice from the Bion-M1 flight showed significantly increased expression of an acinar cell protein, salivary androgen binding protein alpha (SABPα), a pheromone involved in mate selection [33], and the granular convoluted duct cell proteins epidermal growth factor (EGF) and nerve growth factor (NGF) (**Table 1**). PRP, present in acinar cell secretory granules of both sexes, and submandibular gland protein C (SMGC), present in terminal tubule cell secretory granules of female mice, were not significantly different from controls. In the SLG, PSP expression by demilune cells was significantly increased in mice from the space shuttle STS-131 flight, but not in mice from STS-135 [34]. The expression of both PKA-RII in demilune cells, and the acinar cell mucin Muc19,

*Quantitative TEM immunogold labeling of secretory proteins in SMG of female mice flown on STS-131 and* 

*Quantitative TEM immunogold labeling of nuclear PKA-RII in PG of mice flown on STS-131 and STS-135, and the Bion-M1 biosatellite. Labeling results are shown as a percentage of the corresponding habitat control* 

SABPα 114.6 ± 12.1 88.9 ± 26.3 221.9 ± 31.7\* PRP 111.1 ± 13.0 76.6 ± 9.0 134.4 ± 11.5 EGF 87.7 ± 12.4 36.3 ± 6.0 159.9 ± 4.6\* NGF 116.3 ± 23.3 89.9 ± 5.1 159.0 ± 10.6\* SMGC 83.0 ± 9.3 103.4 ± 5.3 —

*Labeling results are shown as a percentage of the corresponding habitat control mice ± SEM.*

**STS-131 STS-135 Bion-M1**

Polyacrylamide gel electrophoresis (PAGE) is the separation of proteins based on mobility on a gel matrix, subjected to a charge differential between two sealed chambers, of a body fluid or tissue extract. This technique is frequently used to determine the distribution or banding pattern of protein groups in tissues under different conditions. Quantification of the intensity of the bands and their relative mobility by PAGE is carried out by densitometric analysis. Saliva is a convenient

**60**

**Figure 6.**

*mice ± SEM.*

*\* p < 0.01.*

**Table 1.**

were increased, but not significantly.

*2.2.2 Electrophoresis and Western blotting*

*STS-135, and male mice flown on the Bion-M1 biosatellite.*

*Electrophoresis and densitometry of saliva from three randomly selected individuals (A, B, and C) from a pool of approximately 50 test subjects. The left ordinate on the gel pattern image shows molecular based migration rates of a mixture of proteins of known size. At the right of the polyacrylamide gel electrophoresis (PAGE) banding patterns are the corresponding densitometric measurements. The top row (−) prior to, the bottom row (+) after, chewing. The asterisks show the migration distance of RII that can be identified by Western blotting.*

body fluid for measuring a variety of physiological conditions or responses on the basis of variations in protein content or distribution. For example, after testing more than 50 samples, PAGE separation of salivary proteins has shown that each individual pattern is unique. Namely, no superimposable patterns were found, therefore constituting essentially a salivary protein pattern fingerprint. Shown at the left in **Figure 7**, in duplicate, are the patterns of three (A, B, C) randomly selected individuals with the corresponding densitometric profiles at the right. **Figure 7** also shows how normal chewing changes the amplitude of several protein peaks, but not the basic pattern.

The PAGE protein banding patterns of the STS mission samples showed a decrease of some of the higher molecular weight bands while the faster moving smaller bands increased when these were compared to vivarium controls. The changes were not consistent between missions and may in part be due to lack of stability of the tissue or of sample preparation.

Western blotting is a technique using antibodies generated to a specific protein for its identification among numerous others in the banding pattern. Proteins are transferred onto nitrocellulose membranes and stained with a washable dye to show the band distribution, then quantified by densitometry. The dye is removed and the membrane probed for reactivity with a specific antibody. These techniques were employed to test salivary gland extracts to measure protein patterns of flight animal samples when compared with those of ground control animals (**Figure 8**).

The results show that the RII peaks in the STS-135 flight samples (**Figure 8**) were significantly smaller than the corresponding band amplitude in either vivarium or habitat controls, middle and bottom profiles, respectively. Western blotting experiments to determine the expression of α-amylase show a significant decrease in the flight and in the habitat controls.

These results indicate that on the shorter, STS-135 flight the expression of RII was decreased, while in the longer Bion-M1 flight (**Figure 9**) an apparent stabilization or adjustment to the microgravity environment had occurred and the RII levels were not different, perhaps even increased from those of either of the controls. Both Bion-M1 and STS-135 flight and both control samples show a significant, but opposite change of a faster moving band that may represent a small protein or peptide.

The findings regarding RII, generally thought to be a stress associated protein [35, 36] responding to the conditions of spaceflight [7], varied by methods of analysis but also between flights within a particular test measure from morphometric analyses of immunogold labeling experiments. Bion-M1 Western blotting results showed no difference between RII in Bion-M1 flight and vivarium control

#### **Figure 8.**

*Densitometric analysis of PAGE protein separation and anti-RII and anti-α-amylase reactivity in STS-135 mice. (A) PG protein samples from mice housed in, top panel, flight; middle panel, habitat; and bottom panel, vivarium type cages. 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. (B) PG α-amylase Western blotting. The ordinate shows integrated density, and the error bars show ±5% error. C1, vivarium control; C2, habitat control; F, flight. The values on the ordinate axis (ID) are integrated density in arbitrary units (with permission, from [13]).*

mice. Measuring RII, therefore, may be a sensitive test for stress reactions during spaceflight.

The variability may be due to the sensitivity of responses affecting RII synthesis. Previous studies point to a rapid and transient increase in the cases of brief acute stress, for example lift off of a spacecraft, followed by long-term, perhaps low level, prolonged time under stress, decrease. For example, a 60-day to a year tour resulting in a decrease of RII and leveling off at concentrations below normal. Eventual physiologic adjustment to flight conditions may start to bring RII production toward normal. An apparent increase also may result from a rough landing that might have occurred in the Bion-M1 flight and the acute response obliterating any reduction due to chronic long-term flight conditions.

Tissue extract samples showed a large faster moving component, presumably an RII fragment. The fragments did resemble the densitometric changes in the flight versus the control. These differences did not completely account for the change of flight values compared to the control. There may have been smaller fragments not visible on the Western blot that could account for the decreases in flight RII.

**63**

**2.3 Gene expression**

*Amy1, α-amylase; Bpifa2, PSP, Prpmp5, PRP; Prkar2a, RII.*

**Figure 10.**

**Figure 9.**

genes were downregulated.

Microarray analyses of the PG showed significant changes in the expression of numerous genes of flight mice compared to habitat ground control mice. In female mice flown on STS-135, the expression of 130 genes was significantly upregulated (log2 ratio ≥ 1), and 75 genes were downregulated (log2 ratio ≤ − 1), (**Figure 10**). In male mice flown on Bion-M1, the expression of 70 genes was upregulated, and 65

*Microarray analysis of secretory protein gene expression in PG of mice flown on STS-135 and the Bion-M1 biosatellite. Results are expressed as log2 of the ratio of expression in flight mice to that in habitat control mice.* 

*Electrophoresis and Western blotting of flight (F) and control (C) PG extracts from mice of the Bion-M1 mission. (A) Protein banding patterns of the flight and control animals (F and C). The left lane (M) shows the relative mobilities of marker proteins of kilodalton (kD) size from 110 to 15 marked on the ordinate. Western blotting for RII is shown in the two right most lanes with two major bands, the first at an estimated 50 kDa size (RII), the second deemed to be a fragment between 14 and 20 kDa (Rfr). (B) densitometric profile of the control banding pattern which was not distinguishable from that of the flight (with permission, from [13]).*

*Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728* *Oral Tissue Responses to Travel in Space DOI: http://dx.doi.org/10.5772/intechopen.86728*

#### **Figure 9.**

*Beyond LEO - Human Health Issues for Deep Space Exploration*

mice. Measuring RII, therefore, may be a sensitive test for stress reactions during

*Densitometric analysis of PAGE protein separation and anti-RII and anti-α-amylase reactivity in STS-135 mice. (A) PG protein samples from mice housed in, top panel, flight; middle panel, habitat; and bottom panel, vivarium type cages. 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. (B) PG α-amylase Western blotting. The ordinate shows integrated density, and the error bars show ±5% error. C1, vivarium control; C2, habitat control; F, flight. The values on the ordinate axis (ID) are integrated density in* 

reduction due to chronic long-term flight conditions.

The variability may be due to the sensitivity of responses affecting RII synthesis. Previous studies point to a rapid and transient increase in the cases of brief acute stress, for example lift off of a spacecraft, followed by long-term, perhaps low level, prolonged time under stress, decrease. For example, a 60-day to a year tour resulting in a decrease of RII and leveling off at concentrations below normal. Eventual physiologic adjustment to flight conditions may start to bring RII production toward normal. An apparent increase also may result from a rough landing that might have occurred in the Bion-M1 flight and the acute response obliterating any

Tissue extract samples showed a large faster moving component, presumably an RII fragment. The fragments did resemble the densitometric changes in the flight versus the control. These differences did not completely account for the change of flight values compared to the control. There may have been smaller fragments not visible on the Western blot that could account for the decreases in flight RII.

**62**

spaceflight.

*arbitrary units (with permission, from [13]).*

**Figure 8.**

*Electrophoresis and Western blotting of flight (F) and control (C) PG extracts from mice of the Bion-M1 mission. (A) Protein banding patterns of the flight and control animals (F and C). The left lane (M) shows the relative mobilities of marker proteins of kilodalton (kD) size from 110 to 15 marked on the ordinate. Western blotting for RII is shown in the two right most lanes with two major bands, the first at an estimated 50 kDa size (RII), the second deemed to be a fragment between 14 and 20 kDa (Rfr). (B) densitometric profile of the control banding pattern which was not distinguishable from that of the flight (with permission, from [13]).*

#### **Figure 10.**

*Microarray analysis of secretory protein gene expression in PG of mice flown on STS-135 and the Bion-M1 biosatellite. Results are expressed as log2 of the ratio of expression in flight mice to that in habitat control mice. Amy1, α-amylase; Bpifa2, PSP, Prpmp5, PRP; Prkar2a, RII.*

#### **2.3 Gene expression**

Microarray analyses of the PG showed significant changes in the expression of numerous genes of flight mice compared to habitat ground control mice. In female mice flown on STS-135, the expression of 130 genes was significantly upregulated (log2 ratio ≥ 1), and 75 genes were downregulated (log2 ratio ≤ − 1), (**Figure 10**). In male mice flown on Bion-M1, the expression of 70 genes was upregulated, and 65 genes were downregulated.

Interestingly, significant changes in gene expression occurred in the PG of habitat ground control mice compared to vivarium housed mice. In ground control female mice housed in the NASA Animal Enclosure Modules, the expression of 50 genes was significantly upregulated, and 33 genes were downregulated. For example, Western blotting of PG of STS-135 habitat control mice showed a decrease in the expression of α-amylase (**Figure 8B**). In ground control male mice housed in the Bion-M1 habitats, the expression of 42 genes was significantly upregulated, and 169 genes were downregulated. These results indicate that the effects of the habitats must be considered when assessing the overall effects of spaceflight on animal (as well as human) physiology.

Changes in the expression of secretory protein genes (**Figure 10**), for example, PKA-RII, largely paralleled the changes seen in protein expression as determined by immunogold labeling and Western blotting (e.g., PKA-RII expression in PG from STS-135 flight mice, **Figure 8B**).

There have been a few previous studies of salivary glands of rodents following spaceflight, and saliva collected from astronauts and cosmonauts before and after spaceflight. No changes in PG morphology were seen by light microscopy in mice flown for 12.5 days on Apollo 17 [37]. The SMG of rats flown for 18.5 days on Cosmos 936 and Cosmos 1129 showed a reduced glycoprotein content as determined by light microscopic histochemical staining [38, 39]. The saliva of 18 astronauts making up the primary and backup crews for Skylab missions of 28-, 59- and 84-days was analyzed preflight and postflight for flow rate, electrolytes, protein, immunoglobulin A (IgA) and lysozyme [40]. Changes were minimal except for an increase in IgA and a decrease in lysozyme concentrations. α-Amylase in cosmonaut saliva was decreased following an 18-day flight (uncited statement in [41]). The use of saliva for monitoring drug absorption was proposed, and saliva levels of acetaminophen were determined in 1 astronaut [42]. Analyses of saliva of cosmonauts during long-term spaceflight showed levels of cortisol below baseline [43], whereas salivary cortisol biorhythms and concentrations were preserved in astronauts onboard Spacelab [44] and cosmonauts on the International Space Station (ISS) [45]. Transient increases in saliva urea and phosphate concentrations, monitored in 2 astronauts during 6 months on the ISS, were consistent with serum concentrations reported in earlier studies [46]. Although studied in astronaut plasma, not saliva, in the NASA Twin Study, EGF and NGF levels were altered during a 340-day flight and after landing [47]. Our studies and those cited above indicate the feasibility of using saliva to monitor astronaut physiology and health.
