Solubilized eggshell membrane supplies a type III collagen-rich elastic dermal papilla.

Signs of aging in facial skin correlate with lifespan and chronic disease; however, the health of aging skin has not been extensively studied. In healthy young skin, the dermis forms a type III collagen-rich dermal papilla, where capillary vessels supply oxygen and nutrients to basal epidermal cells. Chicken eggshell membranes (ESMs) have been used as traditional medicines to promote skin wound healing in Asian countries for many years. Previously, we designed an experimental system in which human dermal fibroblasts (HDFs) were cultured on a dish with a solubilized ESM (S-ESM) bound to an artificial phosphorylcholine polymer; we found that genes that promoted the health of the papillary dermis, such as those encoding type III collagen, were induced in the S-ESM environment. The present study found that a gel with a ratio of 20% type III/80% type I collagen, similar to that of the baby skin, resulted in a higher elasticity than 100% type I collagen (p < 0.05) and that HDFs in the gel showed high mitochondrial activity. Thus, we decided to perform further evaluations to identify the effects of S-ESM on gene expression in the skin of hairless mice and found a significant increase of type III collagen in S-ESM. Picrosirius Red staining showed that type III collagen significantly increased in the papillary dermis after S-ESM treatment. Moreover, S-ESM application significantly improved human arm elasticity and reduced facial wrinkles. ESMs may have applications in extending lifespan by reducing the loss of tissue elasticity through the increase of type III collagen.

Gravitational Effects on Human Physiology.

Physical working capacity decreases with age and also in microgravity. Regardless of age, increased physical activity can always improve the physical adaptability of the body, although the mechanisms of this adaptability are unknown. Physical exercise produces various mechanical stimuli in the body, and these stimuli may be essential for cell survival in organisms. The cytoskeleton plays an important role in maintaining cell shape and tension development, and in various molecular and/or cellular organelles involved in cellular trafficking. Both intra and extracellular stimuli send signals through the cytoskeleton to the nucleus and modulate gene expression via an intrinsic property, namely the "dynamic instability" of cytoskeletal proteins. αB-crystallin is an important chaperone for cytoskeletal proteins in muscle cells. Decreases in the levels of αB-crystallin are specifically associated with a marked decrease in muscle mass (atrophy) in a rat hindlimb suspension model that mimics muscle and bone atrophy that occurs in space and increases with passive stretch. Moreover, immunofluorescence data show complete co-localization of αB-crystallin and the tubulin/microtubule system in myoblast cells. This association was further confirmed in biochemical experiments carried out in vitro showing that αB-crystallin acts as a chaperone for heat-denatured tubulin and prevents microtubule disassembly induced by calcium. Physical activity induces the constitutive expression of αB-crystallin, which helps to maintain the homeostasis of cytoskeleton dynamics in response to gravitational forces. This relationship between chaperone expression levels and regulation of cytoskeletal dynamics observed in slow anti-gravitational muscles as well as in mammalian striated muscles, such as those in the heart, diaphragm and tongue, may have been especially essential for human evolution in particular. Elucidation of the intrinsic properties of the tubulin/microtubule and chaperone αB-crystallin protein complex systems is expected to provide valuable information for high-pressure bioscience and gravity health science.

Control of structural redundancy from the head to trunk in the human upright standing revealed using a data-driven approach.

The human being dynamically and highly controls the head-trunk with redundant mechanical structures to maintain a stable upright standing position that is inherently unstable. The posture control strategies are also affected by the differences in the conditions of sensory inputs. However, it is unclear how the head-trunk segmental properties are altered to respond to situations that require appropriate changes in standing posture control strategies. We used a data-driven approach to conduct a multipoint measurement of head-trunk sway control in a quiet standing position with differences in the conditions of sensory inputs. Healthy young subjects with 22 accelerometers attached to their backs were evaluated for head-trunk vibration during quiet standing under two conditions: one with open eyes and one with closed eyes. The synchronization of the acceleration and the instantaneous phase was then calculated. The results showed that the synchronization of acceleration and instantaneous phase varied depending on the visual condition, and there were some continuous coherent patterns in each condition. Findings were that the structural redundancy of the head-trunk, which is multi-segmental and has a high mass ratio in the whole body, must be adjusted adaptively according to the conditions to stabilize upright standing in human-specific bipeds.

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts.

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

Continuous mild heat stress induces differentiation of mammalian myoblasts, shifting fiber type from fast to slow.

Local hyperthermia has been widely used as physical therapy for a number of diseases such as inflammatory osteoarticular disorders, tendinitis, and muscle injury. Local hyperthermia is clinically applied to improve blood and lymphatic flow to decrease swelling of tissues (e.g., skeletal muscle). As for muscle repair following injury, the mechanisms underlying the beneficial effects of hyperthermia-induced muscle repair are unknown. In this study, we investigated the direct effects of continuous heat stress on the differentiation of cultured mammalian myoblasts. Compared with control cultures grown at 37 degrees C, incubation at 39 degrees C (continuous mild heat stress; CMHS) enhanced myotube diameter, whereas myotubes were poorly formed at 41 degrees C by primary human skeletal muscle culture cells, human skeletal muscle myoblasts (HSMMs), and C2C12 mouse myoblasts. In HSMMs and C2C12 cells exposed to CMHS, mRNA and protein levels of myosin heavy chain (MyHC) type I were increased compared with the control cultures. The mRNA level of MyHC IIx was unaltered in HSMMs and decreased in C2C12 cells, compared with cells that were not exposed to heat stress. These results indicated a fast-to-slow fiber-type shift in myoblasts. We also examined upstream signals that might be responsible for the fast-to-slow shift of fiber types. CMHS enhanced the mRNA and protein levels of peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha in HSMMS and C2C12 cells but not the activities of MAPKs (ERK1/2 and p38 MAPK) in HSMMs and C2C12 cells. These data suggest that CMHS induces a fast-to-slow fiber-type shift of mammalian myoblasts through PGC-1alpha.

Tissue stiffness induced by prolonged immobilization of the rat knee joint and relevance of AGEs (pentosidine).

Joints, connective tissues consisting of extracellular matrix (ECM) with few blood vessels, transfer tension to the skeleton in response to environmental demand. Therefore, joint immobilization decreases active and passive mechanical stress, resulting in increased joint stiffness and tissue degeneration; however, the cause of joint stiffness is obscure. Using a rat knee immobilization model, we examined the relationship between range of motion (ROM) and cell numbers and ECM cross-links by accumulation of advanced glycation end products, pentosidine, in the posterior joint capsule of immobilized joints during 16 weeks of immobilization. The left knee joint was immobilized by internal fixation and compared with the non-immobilized right leg. As early as 2 weeks of immobilization, joint ROM and torque significantly decreased and in parallel, disordered alignment of collagen fiber bundles significantly increased, compared with non-immobilized joints. Those changes continued until 16 weeks of immobilization. Significant increases in pentosidine-positive areas after 8 weeks and significantly decreased cell numbers after 16 weeks of immobilization were also observed compared to the contralateral side. A significant negative correlation between tissue stiffness measured by restriction of ROM and accumulation of pentosidine was observed. This study is the first to show that immobilization of knee joints induces articular contracture associated with sequential changes of ECM alignment, influencing ROM and later pentosidine accumulation and decreased cell numbers during the 16-week immobilization period. Pentosidine appears to be an indicator toward a chronic tissue stiffness leading to decreased cell number rather than a cause of ROM restriction induced by joint immobilization.

Morning Heart Rate Variability as an Indication of Fatigue Status in Badminton Players during a Training Camp.

This study aimed to clarify whether changes in the fatigue status of elite athletes during a precompetition period could be evaluated using morning heart rate variability (HRV) indices. Eight Japanese National Badminton Team players (age, 23.0 ± 2.8 years) participated in this study. HRV and subjective fatigue were measured during the first (days 1-4: Phase 1) and the second half (days 5-8: Phase 2) of an 8-day national team training camp. The global and parasympathetic HRV indices were as follows: standard deviation of all R-R intervals (SDNN) (Phase 1, 87.5 ms; Phase 2, 104.3 ms; p < 0.05), root mean square of the successive R-R interval differences (RMSSD) (Phase 1, 66.6 ms; Phase 2, 103.6 ms; p < 0.05), and high-frequency component power (HF) (Phase 1, 1412.0 ms2; Phase 2, 3318.5 ms2; p < 0.05). All the aforementioned indices increased significantly from Phase 1 to Phase 2. Significant correlations were observed between the change in subjective fatigue and changes in SDNN, RMSSD, and HF (ρ = -0.80, p = 0.017; ρ = -0.77, p = 0.027; and ρ = -0.80, p = 0.017, respectively). Measuring morning HRV indices may be effective for objectively evaluating changes in the fatigue status of elite athletes during a precompetition period.

Changes in Duration and Intensity of the World's Top-Level Badminton Matches: A Consideration of the Increased Acute Injuries among Elite Women's Singles Players.

The purpose of this study was to clarify whether there have been any specific changes in the characteristics of the world's top-level women's singles badminton matches compared to men's singles matches after the current badminton scoring system was implemented in 2006. We compared the characteristics of the matches between the Super Series tournaments in 2007 and 2017. Match duration increased as the rally and rest times increased in both men's and women's singles matches. Specifically, in women's singles, it was suggested that a further increase in physical demands because of the increased number of shots per second may have resulted in longer rest time in proportion to rally time. Moreover, increases in match duration (final eight, 53.3 ± 6.6 min; early rounds, 42.1 ± 3.6 min; P < 0.05) and number of shots per rally (final eight, 10.4 ± 1.2; early rounds, 8.7 ± 1.1; P < 0.05) in women's singles were more prominent in the final eight rounds (quarterfinals, semifinals, and finals) than in the early rounds (rounds 1 and 2). The recent changes in characteristics of the world's top-level badminton matches may account for the increased acute injuries that are frequently observed in elite women's singles players. Thus, appropriate training programs are crucial to effectively improve performance and prevent injuries among elite badminton players.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 µM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

Comparison of Morning Heart Rate Variability at the Beginning and End of a Competition Season in Elite Speed Skaters.

The aim of this study was to clarify whether the physiological fatigue status of elite speed skaters is influenced by the approximately five-month international competition season by comparing morning heart rate variability (HRV) at the beginning of the competition season (Japan Single Distances Championships: JSDC) with that at the end of the competition season (World Single Distances Championships: WSDC). Five international-class speed skaters participated in the study. HRV indices and subjective fatigue were measured each morning of the four days prior to the first races of the JSDC and WSDC in the 2007/2008 season. The parasympathetic HRV indices: root mean square of the successive R-R interval differences (RMSSD) (JSDC, 61.0 ms; WSDC, 42.1 ms; p < 0.05), high-frequency component power (HF) (JSDC, 1393 ms2; WSDC, 443 ms2; p < 0.05), and normalized unit of HF (HFnu) (JSDC, 53.2%; WSDC, 25.5%; p < 0.05) were lower for the WSDC than for the JSDC. The decrease in these indices may reflect the skaters' accumulated fatigue during the course of the competition season. Morning measurements of HRV may thus be an efficient way for elite speed skaters and coaches to objectively monitor physiological fatigue throughout the competition season.

Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation.

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.

Age-related increase of insoluble, phosphorylated small heat shock proteins in human skeletal muscle.

Among mammalian heat shock proteins (Hsps), small Hsps (sHsps) are constitutively expressed in skeletal muscles. We investigated age-related changes of phosphorylation and cellular distribution of representative sHsps (Hsp27 and alphaB-crystallin) in human vastus lateralis muscle under resting conditions. We also examined upstream kinases which may be responsible for phosphorylation of sHsps, namely p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase-2, and extracellular signal-regulated kinase-1/2. The study groups consisted of nine young (15-38 years old) and nine aged (51-79 years old) patients who underwent orthopedic surgery. sHsps protein levels were higher in the insoluble fraction of aged muscles. The phosphorylated states of sHsps were enhanced in both the soluble and insoluble fraction of aged patients. The phosphorylated form of each upstream kinase was elevated in aged patients. Ubiquitinated proteins accumulated in the insoluble fractions of aged muscles. Aging mechanisms may affect the activation process of MAPKs, and the phosphorylation and accumulation of sHsps.

Molecular aspects of adrenal regulation for circadian glucocorticoid synthesis by chronic voluntary exercise.

Chronic voluntary running of mice is known to increase the circadian peak of plasma corticosterone without change in the level of adrenocorticotropic hormone (ACTH). In order to investigate how chronic exercise modulates the circadian HPA axis, we used two weeks of voluntary wheel running of mice and confirmed the significant increase of the circadian peak of plasma corticosterone without alteration in ACTH level. To elucidate the mechanisms of exercise modulation on corticosterone synthesis, we first examined the levels of transcripts involved in corticosterone synthesis of the adrenal gland. Among them, only steroidogenic acute regulatory protein (StAR), the rate-limiting factor that transfers substrate cholesterol into inner mitochondrial membrane, showed significantly higher expression in the exercise group. Since the splanchnic nerve input to the adrenal gland has been reported as a factor involved in the direct modulation of corticosterone synthesis, we next examined the expression levels of enzymes for the catecholamine synthesis as indices of sympatho-adrenomedullary activity. We found that the only rate-limiting enzyme, tyrosine hydroxylase (TH), was significantly higher in the adrenals of exercise group. In addition to the increment of StAR and TH mRNA in response to the chronic exercise, surprisingly, we found only these factors showed the circadian variation in its expression levels that was correlated to the circadian rhythm of corticosterone. Chronic exercise seems to alter the circadian corticosterone synthesis, at least partially via altering the levels of circadian-regulated transcripts, StAR and TH of the adrenal gland.

The free moment is associated with torsion between the pelvis and the foot during gait.

BACKGROUND: During walking, the friction between the foot and the ground surface causes a free moment (FM), which influences the torsional stress on the lower extremity. However, few studies have investigated the FM during natural walking. The main aim of this study was to examine the relationship between the FM and the absolute and relative rotation angles of the foot and pelvis. METHODS: The rotation angles of foot and pelvic were measured in 18 healthy men using a motion capture system. Rotation angles were measured in absolute and relative coordinates as well as in reference to the line connecting the center of pressure (CoP) line under the right and left feet to evaluate the effects of the opposite lower limb on the FM. The absolute and relative rotation angles of the foot and pelvis were entered into forced-entry linear regression models to evaluate the influence on the FM. FINDINGS: Only the relative angle of rotation between the foot and pelvis could explain the prediction equations significantly. In the Pearson's product-moment correlation coefficient, the rotation angles of the foot and pelvis defined using the bilateral CoP points had not significantly correlated with FM. No joint rotation movement was correlated with FM. INTERPRETATION: The torsion of the entire lower extremity should be performed principally through hip internal rotation. When evaluating the FM as a torsional stress, focusing on the rotation of the entire lower extremity, rather than on one segment, is beneficial.

The decrease of the cytoskeleton tubulin follows the decrease of the associating molecular chaperone alphaB-crystallin in unloaded soleus muscle atrophy without stretch.

The cytoskeletal component tubulin/microtubule commonly allows the cell to respond mechanically to the environment. The concentration of free tubulin dimer is autoregulated in the balance of free dimer and polymeric forms of microtubule (MT) protein, having an intrinsic property of "dynamic instability", and through cotranslational beta-tubulin mRNA degradation. Recently, we have demonstrated that alphaB-crystallin is a key molecule of muscle atrophy, since alphaB-crystallin has a chaperone-like-activity that suppresses tubulin aggregation and protects the MT disassembly against both Ca2+ and depolymelizing alkaloid in vitro. Most of the small heat-shock proteins (sHsps), including alphaB-crystallin, are expressed in skeletal muscle. However, no report to date has studied the changes of tubulin/MT during muscle adaptation. Here, we examined changes in tubulin content in rat soleus muscles after hindlimb suspension (HS) with/without passive stretch and the recovery. HS induced rapid decreases of soleus muscle mass, most Hsps (alphaB-crystallin, Hsp90, Hsp70, Hsp27, and p20) and tubulin contents in soleus muscle, while heat-shock cognate 70-kDa protein (Hsc70) did not decrease. Soleus muscle mass, most Hsps, and tubulin were maintained with passive stretch. After 5 days' recovery, the levels of tubulin and Hsps, but not Hsc70, were restored to control levels. The interactions of alphaB-crystallin and tubulin/MT were observed with immunoprecipitation with an anti-alpha-tubulin antibody and taxol-dependent MT assembly. Other sHsps were also associated with alphaB-crystallin and MT, whereas Hsp90 and Hsp70 did not co-precipitate with them. These data imply an interaction and close relationship between alphaB-crystallin and tubulin/MTs in muscle tissues. The amount of mRNA of alphaB-crystallin decreased with the muscle atrophy level, whereas the gene expression level of betaI-tubulin was maintained during HS. This means a significant role of post-transcriptional regulation in tubulin/MT system in muscle adaptation, whereas alphaB-crystallin and most sHsps are regulated at the transcriptional level. Additional functional contribution of alphaB-crystallin to tubulin/MTs during myotube formation was examined using C2C12 myoblast cultured cells, the alphaB-crystallin expression of which was decreased or increased. It indicated the necessity of alphaB-crystallin during microtubule reorganization. In conclusion, tubulin/MTs were revealed to be one of the substrates of alphaB-crystallin, and also serial decreases of alphaB-crystallin and tubulin/MT in early soleus muscle atrophy suggest that the chaperone effect of alphaB-crystallin on the cytoskeleton, which may be also dynamically regulated in the muscle cell, is a key mechanism for muscle adaptation and protection of the atrophy and also muscle differentiation.

Small Heat Shock Protein αB-Crystallin Controls Shape and Adhesion of Glioma and Myoblast Cells in the Absence of Stress.

Cell shape and adhesion and their proper controls are fundamental for all biological systems. Mesenchymal cells migrate at an average rate of 6 to 60 µm/hr, depending on the extracellular matrix environment and cell signaling. Myotubes, fully differentiated muscle cells, are specialized for power-generation and therefore lose motility. Cell spreading and stabilities of focal adhesion are regulated by the critical protein vinculin from immature myoblast to mature costamere of differentiated myotubes where myofibril Z-band linked to sarcolemma. The Z-band is constituted from microtubules, intermediate filaments, cell adhesion molecules and other adapter proteins that communicate with the outer environment. Mesenchymal cells, including myoblast cells, convert actomyosin contraction forces to tension through mechano-responsive adhesion assembly complexes as Z-band equivalents. There is growing evidence that microtubule dynamics are involved in the generation of contractile forces; however, the roles of microtubules in cell adhesion dynamics are not well determined. Here, we show for the first time that αB-crystallin, a molecular chaperon for tubulin/microtubules, is involved in cell shape determination. Moreover, knockdown of this molecule caused myoblasts and glioma cells to lose their ability for adhesion as they tended to behave like migratory cells. Surprisingly, αB-crystallin knockdown in both C6 glial cells and L6 myoblast permitted cells to migrate more rapidly (2.7 times faster for C6 and 1.3 times faster for L6 cells) than dermal fibroblast. On the other hand, overexpression of αB-crystallin in cells led to an immortal phenotype because of persistent adhesion. Position of matured focal adhesion as visualized by vinculin immuno-staining, stress fiber direction, length, and density were clearly αB-crystallin dependent. These results indicate that the small HSP αB-crystallin has important roles for cell adhesion, and thus microtubule dynamics are necessary for persistent adhesion.

Physical performance and soleus muscle fiber composition in wild-derived and laboratory inbred mouse strains.

We compared four inbred mouse strains in their physical performance, measured as a maximal treadmill running time, characteristics of soleus muscle, anatomic character, and growth. The strains used were Mus musculus domesticus [C57BL/6 (B6) and BALB/c], Mus musculus molossinus (MSM/Ms), and Mus spretus. Maximal running time was significantly different among these four mouse strains. Running time until exhaustion was highest in MSM/Ms and lowest in M. spretus. Maximal times for the laboratory mouse strains were nearly identical. Soleus muscle fiber type and cross-sectional area also differed significantly among the species. In particular, M. spretus was significantly different from the other inbred mouse strains. Growth in the wild-derived inbred mice appeared to be complete earlier than in the laboratory mice, and the body size of the wild strains was about half that of the laboratory strains. From these results, we propose that wild-derived inbred mouse strains are useful models for enhancing phenotypic variation in physical performance and adaptability.

Proliferation and differentiation of Xenopus A6 cells under hypergravity as revealed by time-lapse imaging.

Xenopus laevis A6 cells, which are cloned epithelial cells from the Xenopus kidney, differentiate into a dome structure when the cells reach confluence. We investigated the gravitational responses of A6 cellular motility during normal differentiation and differentiation under hypergravity conditions using centrifugation (1-100 x g). Progression to dome formation was analyzed by time-lapse micrography. Dome formation and increased expression of Na(+)/K(+)-adenosine triphosphatase were used as markers of differentiation. Interestingly, a high rate of cellular proliferation was observed at a low level of hypergravity (5 x g). Despite this, there was no difference in the time to dome formation between the control cells at primary cell density and those that differentiated under hyper- or hypogravity conditions. In conclusion, this experiment on amphibian cells revealed that the proliferation of A6 cells was strongly affected by gravity conditions, but the differentiation step appears to be controlled by an intra- or intercellular clock.

The change of HSP47, collagen specific molecular chaperone, expression in rat skeletal muscle may regulate collagen production with gravitational conditions.

It is well known that unloading of skeletal muscle with spaceflight leads skeletal muscle atrophy. However, it remains unclear how the extracellular matrix within the muscle and the connective tissues such as tendon and ligament respond to reduced mechanical load including microgravity, although they have been thought to play important roles in both the transmission of force and the signal transduction between cells and tissues. Type-I collagen and type-IV collagen, both of the major components of extracellular matrix and connective tissues. We focused on change of these collagen synthesis with mechanical load. To obtain an insight into the effects of gravitational changing on the protein metabolism of collagen in skeletal muscle during mechanical unloading, reloading after unloading, we investigated changes in the amount of Heat shock protein 47 (HSP47), has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in rat soleus muscle decreases at 5 days after hindlimb suspension (HS). On the other hand, HSP47 in rat soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. RT-PCR analysis showed HSP47 mRNA decreased with HS earlier, as compared with collagen type-I and type-IV mRNA. From these results, the amount of HSP47 changing by gravitational condition may effect on signal transfers in the primary stage of adaptation and the change of HSP47 expression in skeletal muscle may regulate collagen production with gravitational conditions.

The content of heat shock protein 47 (HSP47), a collagen-specific stress protein, changes with gravitational conditions in skeletal muscle.

It is well known that unloading of skeletal muscle with spaceflight or tail suspension leads rat soleus muscle atrophy. Previously, we reported that one of small heat shock protein (sHSP), alpha B-crystallin shows an early dramatic decrease in atrophied rat soleus muscle (Atomi et al, 1991). In this report, we focused to study the gravitational responses of another HSP, which may be reactive to the gravity. HSP47, a collagen-specific stress protein, has been postulated to be a collagen-specific molecular chaperone localized in the ER (Nagata et al, 1992). Western blot analysis revealed that HSP47 in slow skeletal muscle decreases at 5 days after tail suspension (TS) and increased at 5 days recovery after 10 days of TS as compared with the control level. Hypothetically, HSP47 in slow soleus muscle increases at 5 days after hypergravity (HG) induced by the centrifugation. The content of HSP47 in soleus muscle was strongly affected by gravity conditions.