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Salivary alpha‐amylase (sAA) is an enzyme found in saliva and is considered a noninvasive biomarker for sympathetic nervous system activity. While a wide range of sAA activity in response to stress has been reported in nonhuman primates, the effects of stress on sAA activity in common marmosets are still unknown. We tested the hypothesis that advanced age and cognitive function may have an impact on stress‐related sAA reactivity in marmosets. Thirteen marmosets (nine males and five females) had saliva samples collected during a stressful condition (manual restraint stress) at two different time points, with a 60‐min interval. On the next day, the animals underwent the object recognition test (ORT, a type of cognitive test), and then oral examinations. The animals were categorized into two age groups: old (10–13 years), and very old (15–22 years). Irrespective of age, sAA levels showed a significant difference between T1 (mean 2.07 ± 0.86 U/mL) and T2 samples (mean 1.03 ± 0.67 U/mL), with higher values observed at T1 (p < 0.001). The intra‐assay coefficients of variation (CV) for low and high sAA concentrations were 10.79% and 8.17%, respectively, while the interassay CVs for low and high sAA concentrations were 6.39% and 4.38%, respectively. Oral health issues were common but did not significantly impact sAA levels. The ORT indicated that the animals could recognize an object placed in the cage 6 h after familiarization. In conclusion, all marmosets showed a higher sAA concentration in the first saliva sample as compared to the second saliva sample collected 1 h later, indicating adaptation to stress. No significant differences in sAA levels were observed between sexes, ORT performance, or oral health. Our results indicate that autonomic responsivity and cognitive (memory) functions were preserved even in very old marmosets. Highlights: This study examines how advanced age and cognitive function impact salivary alpha‐amylase (sAA) reactivity.sAA, a stress biomarker, varies in response to stress in common marmosets, largely unexplored till now.Results show higher sAA levels during initial stress, with preserved autonomic and cognitive function in older marmosets. [ABSTRACT FROM AUTHOR]

Background: Non‐human primates are the closest animal models to humans regarding genetics, physiology and behaviour. Marmoset monkeys in particular are one of the most versatile species for biomedical research. Objective: To assess the craniofacial growth and development of the masticatory function in the common marmoset (Callithrix jacchus), from birth to the fourth month of life through minimally invasive cineradiographic imaging. Methods: Ten individuals were followed‐up from 0 to 4 months of age regarding craniofacial growth and masticatory function assessed by cineradiography. For the experimental procedure, we used a microfocal X‐ray source apparatus and a beryllium fast‐response image‐intensifier. Results: The duration of the masticatory cycles was stable across age groups. Chewing a very soft Castella cake or the slightly harder Marshmallow did not change the masticatory cycle in the time domain. On the other hand, linear and angular measurements of the jaw‐opening movement showed a tendency for bigger movements at the latter stages of craniofacial growth. Qualitative analysis showed that marmosets had a small preference for Castella over Marshmallow, that they most often bit off pieces of food to chew with their posterior teeth, that they manipulated the food with their hands, and that they chewed the food continuously. Conclusion: We observed critical developmental events during the first 3 months of life in marmosets. Cineradiographic imaging in marmosets may provide valuable information on craniofacial form and function for basic and preclinical research models. [ABSTRACT FROM AUTHOR]

With rapid aging of the world’s population, the demand for research, for a better understanding of aging and aging-related disorders, is increasing. Ideally, such research should be conducted on human subjects. However, due to ethical considerations, animals such as rodents and monkeys are used as alternatives. Among these alternative models, non-human primates are preferred because of their similarities with humans. The small South American common marmoset (Callithrix jacchus) may offer several advantages over other non-human primates in terms of its smaller size, shorter life-span, and dental anatomy identical to humans. The purpose of this study was to determine the viability of using the marmoset as a human oral disease model. We collected saliva samples from eight marmosets and eight human subjects. Prokaryotic DNA was extracted from the saliva samples, and 16S bacterial rRNA gene sequencing was performed on each of the samples. Our results indicated that the types of oral microbiomes detected among human and marmoset samples were nearly indistinguishable. In contrast, the oral microbiomes of our human and marmoset subjects were distinctly different from those reported for rats and dogs, which are currently popular research animals. The oral microbiomes of marmosets showed greater diversity than those of humans. However, the oral microbiota of marmosets exhibited less variation than those of humans, which may be attributed to the fact that the marmoset subjects were kept in a controlled environment with identical lifestyles. The characteristics of its oral microbiota, combined with other technical advantages, suggest that the marmoset may provide the best animal model thus far for the study of oral health. This study characterized the oral microbes of the marmoset, thereby providing information to support future application of the marmoset as a model for age-related oral disease. [ABSTRACT FROM AUTHOR]

Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized after a 24-h treatment with 1α,25 dihydroxyvitamin D3. The quantitative RT-PCR determined the mRNA levels of signaling molecules upstream and downstream Ras. The small GTPase is activated by guanine nucleotide exchange protein (GEF) and deactivated by GTPase-activating protein (GAP). When external stimuli are transduced into intracellular signals, various pathways are recruited: focal adhesion kinase (FAK) is associated with integrin-β, and directs tyrosine phosphorylation of downstream substrates, including phospholipase C-γ (PLC-γ) and son of sevenless (SOS, a Ras GEF). The mRNA levels of FAK and PLC-γ1 and -γ2 in the flight cultures were increased 150% and 250% of the ground controls. The SOS mRNA levels in the flight cultures were increased 520% and 320% of the ground controls. Signals via G protein–coupled receptors are transmitted through PLC-β and Ras GRF (another Ras GEF). Activated Ras then stimulates Raf, mitogen-activated protein kinase (MAPK) cascades. The mRNA levels of Raf, extracellular signal-regulated protein kinase of MAPK family (ERK-1 and -2), and PLC-β were increased during spaceflight. Rho GAP expression in the flight cultures was increased twofold of the ground controls. Since Rho GAP deactivates Rho, microgravity may suppress Rho signals, regulating actin filament rearrangement. Microgravity signals may involve two pathways (G protein–coupled receptor-mediated pathway and tyrosine phosphorylation-mediated pathway) that activate Ras, Raf, and MAPK cascades in rat osteoblasts. [ABSTRACT FROM AUTHOR]

Rat osteoblasts were cultured for 4 or 5 days aboard the Space Shuttle and solubilized during spaceflight. Post-flight analyses by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) determined the relative mRNA levels of matrix proteins, adhesion molecules, and cytoskeletal proteins including osteopontin (OP), osteonectin (ON), CD44, α-tubulin, actin, vimentin, fibronectin (FN), and β1-integrin. The mRNA levels of OP and α-tubulin in the flight cultures were decreased by 30% and 50% on day 4 and day 5 of flight, as compared to the ground controls. In contrast, the CD44 mRNA levels in the flight cultures increased by 280% and 570% of the ground controls on day 4 and day 5. The mRNA levels of ON and FN in the flight cultures were slightly increased as compared to ground controls. The mRNA levels of actin, vimentin, or β1-integrin did not change in spaceflight conditions. The matrix proteins, adhesion molecules, and cytoskeletal proteins may form dynamic network complexity with signaling molecules as an adaptive response to perturbation of mechanical stress under microgravity. [ABSTRACT FROM AUTHOR]

Physiological actions of osteoblasts are disordered by gravity unloading. We investigated the possibility that the appropriate level of hypergravity could improve osteoblast functions that are susceptible to mechanical unloading. We evaluated hypergravity effects on the 1α,25-dihydroxyvitamin D3 (VD)-inducible osteocalcin expression of primary rat osteoblasts. Cell culture plates were centrifuged for 24 h at 3, 6, 12, 24, and 48g in a 37°C incubator. The mRNA levels were analyzed by quantitative RT-PCR. The mRNA levels for osteocalcin and vitamin D receptor (VD-R) at 12g were enhanced to 187% and 228% of the 1g control, respectively. However, the excess hypergravity conversely decreased osteocalcin expression. Osteocalcin gene expression was enhanced by VD/VD-R through the vitamin D-responsive element in the promoter. The increased osteocalcin expression might reflect the augmented VD-R expression. Alternatively, Runx2, a master gene of osteoblast differentiation, might be responsible for the osteocalcin induction, since the Runx2 mRNA levels were also increased to 247% of control at 12g. Another VD-inducible osteoblast phenotype, alkaline phosphatase, was also upregulated at 12g and 24g. The appropriate level of hypergravity enhanced the VD-inducible expression of osteocalcin, a typical phenotype of osteoblast differentiation. These data suggest molecular features to prevent disuse bone atrophy of long-term bed-rest patients. [ABSTRACT FROM AUTHOR]

Rat osteoblasts were cultured aboard a space shuttle for 4 and 5 days. Cells were treated with 1 nM 1α,25-dihydroxyvitamin D3 (VD) for the last 1 day. The conditioned media were harvested. Cells were solubilized with guanidine solution on board. We examined microgravity effects on the production/ expression of osteocalcin, bone sialoprotein (BSP), and VD receptor (VD-R) in osteoblasts. Under VD treatment, the osteocalcin protein level was 243 ± 117 and 1,718 ± 534 pg/μg cellular DNA in flight cultures and ground controls, respectively. Without VD treatment, the osteocalcin protein level was not different between flight cultures and ground controls. The osteocalcin mRNA level in the VD-treated flight cultures was as low as 16% of that in ground controls. The VD-R mRNA level in the VD-treated flight cultures was also decreased to 16% of that in ground controls. Microgravity would suppress the VD-inducible production of osteocalcin but not the basal productivity. The BSP mRNA level was increased by microgravity. VD/VD-R binds to the vitamin D-responsive element (VDRE) on the target genes. The rat osteocalcin gene is positively regulated via "enhancer" VDRE, whereas the rat BSP gene is negatively regulated via "repressor " VDRE. Microgravity might modulate osteoblast responsiveness to VD through the suppression of VD-R. [ABSTRACT FROM AUTHOR]

The platelet-activating factor (PAF) is a lipid mediator. The Gprotein- coupled receptor of PAF (PAF-R) is activated by inflammatory and stressful conditions in numerous cell types. PAF/PAF-R is involved in apoptotic and antiapoptotic processes. We examined microgravity effects on the expression of PAF-R and second messengers in rat osteoblasts. The PAF-R signals are transmitted via arachidonic acid, phospholipase C (PLC), protein kinase C (PKC), and mitogen-activated protein kinase. Rat osteoblasts were cultured for 4 and 5 days aboard a space shuttle and solubilized on board. PAF-R gene expression in flight cultures increased to 2-6-fold higher than in ground controls. Gene expression of the G-protein α subunit Gαq in flight cultures increased to 3-fold and higher than in ground controls. It is known that Gαq stimulates the effecter PLCβ , activating PKC. The mRNA levels of PKCδ and PKCν in flight cultures were increased to 2-5-fold higher than in ground controls. The PKCα mRNA level in flight cultures was increased to 3-fold higher than in ground controls on the 4th day. Gene expression of catalytic and regulatory subunits of protein kinase A was suppressed in flight cultures. PKCδ and PKCν are novel PKCs that can be target substrates of caspases. The PAF-R gene may act as a mechano-sensitive gene that is involved in the apoptotic and antiapoptotic processes of osteoblasts under microgravity. [ABSTRACT FROM AUTHOR]

It has been reported that stress exposure could suppress pain in various animal species. However, very few studies have been performed on the effect of gravity stimulation on nociceptors. The aim of the present study is to reveal the effect of gravity stimulation on nociceptive withdrawal responses in rats. A von Frey type apparatus was used to evoke withdrawal reflexes at eight different body sites. Reflex thresholds were measured before and after gravity stimulation, which was applied with a centrifugal-type apparatus at 116 r.p.m. (2 g), for 10 min. Naloxone-HCl (0.1 mg kg[sup -1] i.p.) was used before centrifugal stimulation to investigate whether the analgesic effect was mediated via endogenous opioids. The mean threshold values significantly increased in all body sites after 2.0 g exposure. Moreover, the analgesic effect was greater in upper body sites. The increase in threshold remained long after gravity stimulation, suggesting that humoral factors are involved in the analgesic mechanism. Gravity-induced analgesia was antagonized by naloxone, indicating that an opioid system is included in its mediation. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The mechanism underlying space flight-induced osteopenia is unknown. In osteoblasts, the inducible nitric oxide (NO) synthase (iNOS) is involved in the early response to mechanical strain and induction of apoptosis. GTP cyclohydrolase I (GTPCH) is a key enzyme that is essential for iNOS activity. The coordinate expression of GTPCH prevents apoptosis that is induced by iNOS/NO. The purpose of this study was to investigate the effects of space flight on the expression of apoptotic/anti-apoptotic molecules iNOS and GTPCH in rat osteoblasts. Rat osteoblasts were cultured aboard a space shuttle and solubilized on the 4th and 5th days of the mission. The mRNA levels for iNOS and GTPCH in the flight cultures were increased to at least 120-fold and threefold higher than the ground (1×g) controls, respectively. The amount of cellular DNA per flight culture vessel was 53% and 58% of the ground controls on the 4th and 5th days, respectively. However, the increasing rate of the DNA amount from the 4th to the 5th day was not different between the flight cultures and the ground controls. Morphologically, the cells grew in space as well as on the ground. Co-expression of GTPCH and iNOS may indicate a self-protective mode of action in osteoblasts against the harmful stress under microgravity. [ABSTRACT FROM AUTHOR]

Rat osteoblasts were cultured aboard a space shuttle for 4 or 5 days. Cells were exposed to 1α, 25 dihydroxyvitamin D3 during the last 20 h and then solubilized by guanidine solution. The mRNA levels for molecular chaperones were analyzed by semi-quantitative RT-PCR. ELISA was used to quantify TGF-β1 in the conditioned medium. The HSP70 mRNA levels in the flight cultures were almost completely suppressed, as compared to the ground (1×g) controls. The inducible HSP70 is known as the major heat shock protein that prevents stress-induced apoptosis. The mean mRNA levels for the constitutive HSC73 in the flight cultures were reduced to 69%, ∼ 60% of the ground controls. HSC73 is reported to prevent the pathological state that is induced by disruption of microtubule network. The mean HSP47 mRNA levels in the flight cultures were decreased to 50% and 19% of the ground controls on the 4th and 5th days. Concomitantly, the concentration of TGF-β1 in the conditioned medium of the flight cultures was reduced to 37% and 19% of the ground controls on the 4th and 5th days. HSP47 is the collagen-specific molecular chaperone that controls collagen processing and quality and is regulated by TGF-β1. Microgravity differentially modulated the expression of molecular chaperones in osteoblasts, which might be involved in induction and/or prevention of osteopenia in space. [ABSTRACT FROM AUTHOR]

A functional disorder associated with weightlessness is well documented in osteoblasts. The apototic features of this disorder are poorly understood. Harmful stress induces apoptosis in cells via mitochondria and/or Fas. The Bax triggers cytochrome c release from mitochondria, which can be blocked by the Bcl-2. Released cytochrome c then activates the initiator caspase, caspase-9, which can be blocked by the anti-apototic (IAP) family of molecules. The effector caspase, caspase-3, finally exerts DNA fragmentation. We conducted this study to examine the apoptotic effects of vector-averaged gravity on normal human osteoblastic cells. Cell culture flasks were incubated on the clinostat, which generated vector-averaged gravity condition (simulated microgravity) for 12, 24, 48, and 96 hours. Upon termination of clinostat cultures, the cell number and cell viability were assessed. DNA fragmentation was analyzed on the agarose-gel electrophoresis. The mRNA levels for Bax, Bcl-2, XIAP, and caspase-3 genes were analyzed by semi-quantitative RT-PCR. Twenty-four hours after starting clinostat rotation, the ratios of Bax/Bcl-2 mRNA levels (indicator of apoptosis) were significantly increased to 136% of the 1G static controls. However, the XIAP mRNA levels (anti-apoptotic molecule) were increased concomitantly to 138% of the 1G static controls. Thus, cell proliferation or cell viability was not affected by vector-averaged gravity. DNA fragmentation was not observed in clinostat group as well as in control group. Finally, the caspase-3 mRNA levels were not affected by vector-averaged gravity. Simulated microgravity might modulate some apoptotic signals upstream the mitochondrial pathway. [ABSTRACT FROM AUTHOR]

A three-dimensional (3D) clinostat is a device for multidirectional G force generation. By controlled rotation of two axes, a 3D clinostat cancels the cumulative gravity vector at the center of the device and produces an environment with an average of 10−3 G over time. We cultured a human osteoblast cell line in a 3D clinostat and examined the growth properties and differentiation of the cells, including morphology, histological detection of calcification, and mitogenactivated protein kinase (MAPK) cascades. In a normal 1G condition, alkaline phosphatase (AIPase) activity was detected on day 7 of culture, bone nodules were formed on day 12, and calcium deposits were seen on day 20. In the 3D clinostat, the cell looked larger and bulged. AIPase activity was detected on day 10 of culture. However, neither bone nodules nor calcification was found in the 3D clinostat up to day 21. The expression levels of core-binding factor A1 (a transcription factor for bone formation) and osteocalcin (a bone matrix protein) increased in the control culture but decreased in culture in 3D clinostat. Phosphorylation of p38MARK (p38) was repressed in culture in 3D clinostat, whereas total p38 as well as total and phosphorylated forms of extracellular signal-regulated kinases and stress-activated protein kinase/jun N-terminal kinase were not changed in the 3D clinostat. When a p38 inhibitor, SB 203580, was added to the culture medium in a normal 1 G environment, AIPase activity and formation of bone nodules and calcium deposits were strongly inhibited. On the other hand, they were inhibited only partially by a MARK kinase inhibitor, U-0126. On the basis of these results, it is concluded that (1) osteoblast differentiation is inhibited in culture in a 3D clinostat and (2) this inhibition is mainly due to the suppression of p38 phophorylation. [ABSTRACT FROM AUTHOR]

A number of studies have indicated that microgravity induces osteopenia and modulates functions of mammalian cells. However, the molecular mechanisms underlying these effects of microgravity are still unknown. Rat osteoblasts were cultured for 4 and 5 days during Shuttle-Spacelab flight, and fixed by guanidine isothiocyanate solution on board after treatment with 1α, 25 (OH)2 vitamin D3. The mRNA levels for platelet-derived growth factor (PDGF)-β receptor, epidermal growth factor (EGF) receptor, the growth factor receptor adaptor protein Shc, and c-fos were determined using the method of quantitative reverse transcription-polymerase chain reaction. The mRNA levels for EGF receptor were not altered by microgravity. However, the mRNA levels for PDGF-β receptor, Shc, and c-fos were decreased to 62, 55 and 25% on the 4th day of flight, and 47, 40, and 43% on the 5th day, respectively, as compared to the corresponding ground controls. Expression of the growth factor receptor and the receptor adaptor protein was modulated in rat osteoblasts during spaceflight. Data suggest that signal transduction via growth factor receptors in rat osteoblasts is impaired by microgravity. Dysfunction of osteoblasts might be involved in spaceflight-induced osteopenia. [ABSTRACT FROM AUTHOR]