Establishment and characterization of salivary gland-specific injury in transgenic mice model.

INTRODUCTION: Many mouse models for autoimmune diseases also have lesions in non-target organs, which may make it difficult to determine whether the target organ lesion is primary or secondary. Hyposalivation has conventionally been studied using genetically modified mouse models for Sjogren's syndrome as well as spontaneous autoimmune mice with systemic lesions, none of which has salivary gland-specific injury. METHODS: In this study, we established a salivary gland-specific injury mouse model using the TRECK system by gene modification with the transgene composed of the 5' untranslated region of human salivary mucin gene MUC7 (highly expressed specifically in human salivary gland) inserted at the upstream of hHB-EGF (diphtheria toxin receptor) in the TRECK vector. RESULTS: In this transgenic mouse model, we confirmed salivary gland-specific expression of hHB-EGF gene, and hyposalivation after treatment with diphtheria toxin. Histological assessment of the salivary gland from these mice showed granular convoluted tubule epithelial cells destruction at the same position as a positivity in TUNEL assay. CONCLUSION: This transgenic mouse model may become a useful tool for elucidating the mechanisms involved in hyposalivation and for developing pharmaceuticals and tissue regenerative medical products for this condition.

Effects of polyphenols in non-centrifugal cane sugar on saliva secretion: in vitro and in vivo experiments and a randomized controlled trial.

This study examined the bioactivities and mechanisms of the non-centrifugal cane sugar polyphenols saponarin, schaftoside, and isoschaftoside in the salivary gland and their effects on salivation. In acute isolated C57BL/6N mouse submandibular gland cells, these polyphenols led to a higher increase in intracellular calcium after stimulation with the muscarinic agonist carbachol. Stimulation of these cells with polyphenols enhanced ATP production, aquaporin-5 translocation to the plasma membrane and eliminated intracellular reactive oxygen species generated by H2O2. In addition, phosphorylation of endothelial nitric oxide synthase and increased nitric oxide production in vascular endothelial cells were observed. In vivo administration of these polyphenols to C57BL/6N male mice resulted in significantly increased blood flow (saponarin, p = 0.040; isoschaftoside, p = 0.010) and salivation (saponarin, p = 0.031). A randomized controlled trial showed that intake of non-centrifugal cane sugar significantly increased saliva secretion compared with placebo (p = 0.003). These data suggest that non-centrifugal cane sugar polyphenols affect several pathways that support salivation and increase saliva secretion by enhancing vasodilation. Hence, non-centrifugal cane sugar polyphenols can be expected to maintain saliva secretion and improve reduced saliva flow.

Expanding on the professional role of dental hygienists as key managers of medical-dental and hospital-dental clinic collaboration in a local Japanese hospital without a dentistry department: From a questionnaire survey after a web seminar.

INTRODUCTION: Perioperative oral management (POM) was introduced into the Japanese universal health insurance system in 2012. Collaboration with dental clinics is important for hospitals without a dentistry department. A dental hygienist newly assigned as a member of the patient flow management centre led a seminar to promote collaboration via the web. This study represents the first step to explore the possible role of the hospital-based dental hygienist in the field of regional medical-dental cooperation of POM by assessing their willingness to participate in providing this type of care by a survey. METHODS: The rate of attendees' satisfaction and the current problems of the collaboration for POM were evaluated through a questionnaire survey after the web seminar. RESULTS: All respondents reported satisfaction with the web seminar although it was the first experience of an online seminar for half of the respondents. All hospital dentists, but only 47.8% of dentists working at clinics, had participated in POM. Dental hygienist tended to show greater desire to participate in POM than dentists. All respondents appreciated the role played by the dental hygienist as a key manager of medical-dental collaboration between the hospital and local clinics. CONCLUSION: The hospital-based dental hygienist can play a role in planning and management of web seminars for POM, to raise awareness and promote regional medical-dental cooperation of POM.

Endoplasmic reticulum stress affects mouse salivary protein secretion induced by chronic administration of an α1-adrenergic agonist.

Stress stimulates both the sympathetic-adrenomedullary and hypothalamus-pituitary-adrenal axes. Activation of these axes results in the release of catecholamines, which in turn affects salivary secretion. Thus, repetitive stimulation of the α1-adrenergic receptor could be useful for studying the effects of chronic stress on the salivary gland. Salivary protein concentration and kallikrein activity were significantly lower in mice following chronic phenylephrine (PHE) administration. Chronic PHE administration led to significantly increased expression of the 78-kDa glucose-regulated protein, activating transcription factor 4, and activating transcription factor 6. Histological analyses revealed a decrease in the size of the serous cell and apical cytoplasm. These results suggest that repetitive pharmacological stimulation of the sympathetic nervous system elicits ER stress and translational suppression. In addition, PHE-treated mice exhibited a decrease in intracellular Ca2+ influx elicited by carbachol, a muscarine receptor agonist in the submandibular gland. The present findings suggest that chronic psychological, social, and physical stress could adversely affect Ca2+ regulation.

Possible Action of Olaparib for Preventing Invasion of Oral Squamous Cell Carcinoma In Vitro and In Vivo.

Despite recent advances in treatment, the prognosis of oral cancer remains poor, and prevention of recurrence and metastasis is critical. Olaparib is a PARP1 inhibitor that blocks polyADP-ribosylation, which is involved in the epithelial-mesenchymal transition (EMT) characteristic of tumor recurrence. We explored the potential of olaparib in inhibiting cancer invasion in oral carcinoma using three oral cancer cell lines, HSC-2, Ca9-22, and SAS. Olaparib treatment markedly reduced their proliferation, migration, invasion, and adhesion. Furthermore, qRT-PCR revealed that olaparib inhibited the mRNA expression of markers associated with tumorigenesis and EMT, notably Ki67, Vimentin, ß-catenin, MMP2, MMP9, p53, and integrin α2 and ß1, while E-Cadherin was upregulated. In vivo analysis of tumor xenografts generated by injection of HSC-2 cells into the masseter muscles of mice demonstrated significant inhibition of tumorigenesis and bone invasion by olaparib compared with the control. This was associated with reduced expression of proteins involved in osteoclastogenesis, RANK and RANKL. Moreover, SNAIL and PARP1 were downregulated, while E-cadherin was increased, indicating the effect of olaparib on proteins associated with EMT in this model. Taken together, these findings confirm the effects of olaparib on EMT and bone invasion in oral carcinoma and suggest a new therapeutic strategy for this disease.

Hyperglycemia Induces Generation of Reactive Oxygen Species and Accelerates Apoptotic Cell Death in Salivary Gland Cells.

INTRODUCTION: Type-2 diabetes mellitus (T2DM) is associated with several systemic vascular symptoms and xerostomia. It is considered that hyperglycemia-induced polyuria and dehydration cause decreased body-water volume, leading to decreased saliva secretion and, ultimately, xerostomia. In T2DM, increased production of reactive oxygen species (ROS) causes tissue damage to vascular endothelial cells as well as epithelial tissue, including pancreas and cornea. Hence, a similar phenomenon may occur in other tissues and glands in a hyperglycemic environment. METHODS: Salivary gland tissue injury was examined, using T2DM model mouse (db/db). Transferase-mediated dUTP nick-end labeling (TUNEL) was conducted to evaluate tissue injury. The levels of malondialdehyde (MDA) and 8-hydroxy-2'-deoxyguanosine, Bax/Bcl-2 ratio were measured as indicator of oxidative stress. Moreover, in vitro ROS production and cell injury was evaluated by mouse salivary gland-derived normal cells under high-glucose condition culture. RESULTS: In vivo and in vitro analysis showed a higher percentage of TUNEL-positive cells and higher levels of MDA and 8-hydroxy-2'-deoxyguanosine in salivary gland tissue of db/db mice. This suggests damage of saliva secretion-associated lipids and DNA by hyperglycemic-induced oxidative stress. To analyze the mechanism by which hyperglycemia promotes ROS production, mouse salivary gland-derived cells were isolated. The cell culture with high-glucose medium enhanced ROS production and promotes apoptotic and necrotic cell death. CONCLUSION: These findings suggest a novel mechanism whereby hyperglycemic-induced ROS production promotes salivary gland injury, resulting in hyposalivation.

Pulmonary activation of vitamin D3 and preventive effect against interstitial pneumonia.

Calcitriol [1,25(OH)2D3] is usually investigated in studies on the preventive effect of activated vitamin D against interstitial pneumonia. Although cholecalciferol (vitamin D3) can be easily obtained in the diet and has a longer half-life than calcitriol, there have been few investigations of its effect on interstitial pneumonia. We used human pulmonary fibroblast cell lines (HPFCs) and a mouse model of bleomycin-induced pulmonary fibrosis to evaluate whether vitamin D3 was activated in the lungs and had a preventive effect against interstitial pneumonia. Expression of the vitamin D receptor gene and genes for enzymes metabolizing vitamin D was evaluated in two HPFCs, and the suppressive effect of vitamin D3 on induction of inflammatory cytokines was also assessed. Gene expression of the vitamin D receptor and vitamin D-metabolizing enzymes was observed in both human pulmonary fibroblast cell lines. Vitamin D3 suppressed bleomycin-induced expression of inflammatory cytokines and fibrosis markers by the HPFCs. In mice, symptoms of bleomycin-induced pulmonary fibrosis were improved and expression of fibrosis markers/fibrosis inducers was decreased by a high vitamin D3 diet. Vitamin D3 is activated locally in lung tissues, suggesting that high dietary intake of vitamin D3 may have a preventive effect against interstitial pneumonia.

Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo.

Cisplatin is a commonly used chemotherapeutic drug for treatment of oral carcinoma, and combinatorial effects are expected to exert greater therapeutic efficacy compared with monotherapy. Poly(ADP-ribosyl)ation is reported to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, and genomic stability. Based on these properties, poly(ADP-ribose) polymerase (PARP) inhibitors are used for treatment of cancers, such as BRCA1/2 mutated breast and ovarian cancers, or certain solid cancers in combination with anti-cancer drugs. However, the effects on oral cancer have not been fully evaluated. In this study, we examined the effects of PARP inhibitor on the survival of human oral cancer cells in vitro and xenografted tumors derived from human oral cancer cells in vivo. In vitro effects were assessed by microculture tetrazolium and survival assays. The PARP inhibitor AZD2281 (olaparib) showed synergetic effects with cisplatin in a dose-dependent manner. Combinatorial treatment with cisplatin and AZD2281 significantly inhibited xenografted tumor growth compared with single treatment of cisplatin or AZD2281. Histopathological analysis revealed that cisplatin and AZD2281 increased TUNEL-positive cells and decreased Ki67- and CD31-positive cells. These results suggest that PARP inhibitors have the potential to improve therapeutic strategies for oral cancer.

Resveratrol improves salivary dysfunction in a non-obese diabetic (NOD) mouse model of Sjögren's syndrome.

Resveratrol is a natural polyphenol produced by plants in response to environmental stress. This compound has been shown to have pharmacological effects against a wide range of diseases including neurological, hepatic, cardiovascular and autoimmune conditions. The non-obese diabetic (NOD) mouse, in which loss of lacrimal and salivary gland function occurs, has been studied as an animal model for Sjögren's syndrome. In this study, we confirmed that administration of resveratrol results in increased secretion of saliva in NOD mice. Although resveratrol enhanced Sirt1 activity, inflammatory cell infiltration was not affected. Moreover, expression of the anti-inflammatory cytokine IL-10 in salivary glands was enhanced in the resveratrol-administered group. Thus, we confirmed a novel therapeutic effect for resveratrol on salivary dysfunction in Sjögren's syndrome.

PARP Inhibitor PJ34 Suppresses Osteogenic Differentiation in Mouse Mesenchymal Stem Cells by Modulating BMP-2 Signaling Pathway.

Poly(ADP-ribosyl)ation is known to be involved in a variety of cellular processes, such as DNA repair, cell death, telomere regulation, genomic stability and cell differentiation by poly(ADP-ribose) polymerase (PARP). While PARP inhibitors are presently under clinical investigation for cancer therapy, little is known about their side effects. However, PARP involvement in mesenchymal stem cell (MSC) differentiation potentiates MSC-related side effects arising from PARP inhibition. In this study, effects of PARP inhibitors on MSCs were examined. MSCs demonstrated suppressed osteogenic differentiation after 1 µM PJ34 treatment without cytotoxicity, while differentiation of MSCs into chondrocytes or adipocytes was unaffected. PJ34 suppressed mRNA induction of osteogenic markers, such as Runx2, Osterix, Bone Morphogenetic Protein-2, Osteocalcin, bone sialoprotein, and Osteopontin, and protein levels of Bone Morphogenetic Protein-2, Osterix and Osteocalcin. PJ34 treatment also inhibited transcription factor regulators such as Smad1, Smad4, Smad5 and Smad8. Extracellular mineralized matrix formation was also diminished. These results strongly suggest that PARP inhibitors are capable of suppressing osteogenic differentiation and poly(ADP-ribosyl)ation may play a physiological role in this process through regulation of BMP-2 signaling. Therefore, PARP inhibition may potentially attenuate osteogenic metabolism, implicating cautious use of PARP inhibitors for cancer treatments and monitoring of patient bone metabolism levels.

Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine.

The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.

Aryl hydrocarbon receptor-mediated induction of EBV reactivation as a risk factor for Sjögren's syndrome.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a variety of biological effects by binding to environmental pollutants, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). Although numerous animal studies have demonstrated the harmful effects of dioxins, it remains controversial whether dioxins pose a risk to human health. Enhanced lytic replication of EBV is a risk factor for the development of autoimmune diseases and cancers. This study evaluated the possibility that ligand-activated AhR reactivates EBV. EBV reactivation and AhR transactivation were evaluated with luciferase assays. Saliva samples were collected from 19 patients with primary Sjögren's syndrome (SS). Control saliva samples were obtained from 10 healthy individuals and nine patients with severe dry mouth. TCDD enhanced BZLF1 transcription, which mediates the switch from the latent to the lytic form of EBV infection in EBV-positive B cell lines and in a salivary gland epithelial cell line. Moreover, TCDD-induced increases in BZLF1 mRNA and EBV genomic DNA levels were confirmed in the B cell lines. Saliva from SS patients activated the transcription of both CYP1A1 and BZLF1. Additionally, there was a positive correlation between CYP1A1 and BZLF1 promoter activities. AhR ligands elicited the reactivation of EBV in activated B cells and salivary epithelial cells, and these ligands are involved in SS. Our findings reveal novel aspects of the biological effects of dioxin and the AhR-dependent pathogenesis of autoimmune diseases.

Evaluation of the benefits of neutral bicarbonate ionized water baths in an open-label, randomized, crossover trial.

We previously demonstrated that neutral bicarbonate ionized water (NBIW) bathing enhances blood flow by bicarbonate ions and described the underlying mechanism. However, additional clinical investigation was warranted to investigate the efficacy of NBIW bathing. Hence, we performed a randomized, open-label, crossover trial to examine the effects of NBIW bathing on mental stress, sleep, and immune function. Participants who regularly felt stressed were randomly assigned to NBIW or regular bathing for 4 weeks. Mental stress was assessed with the Brief Job Stress Questionnaire (BJSQ) and the Profile of Mood States Second Edition; sleep quality, with the Pittsburgh Sleep Quality Index Japanese version (PSQI-J) and actigraphy; and immune function, with laboratory tests. PSQI-J scores and actigraphy sleep latency and bed out latency improved significantly more with NBIW bathing than with regular bathing (p < 0.05). Furthermore, NBIW bathing reduced both stress-induced fluctuations in CD4+ and CD8+ T cell counts and fluctuations in the naive to memory T cell ratio and neutrophil phagocytosis, indicating improved immune function. These findings suggest that daily NBIW bathing could improve mental stress, sleep quality, and immune function and bring about positive health effects in those who experience stress in their daily lives.

Characterization of rice endosperm-derived antidepressant-like peptide (REAP): An orally active novel tridecapeptide derived from rice protein.

It is ideal to ingest bioactive substances from daily foods to stay healthy. Rice is the staple food for almost half of the human population. We found that an orally administered enzymatic digest of rice endosperm protein exhibits antidepressant-like effects in the tail suspension test (TST) using mice. A comprehensive peptide analysis of the digest using liquid chromatography-tandem mass spectrometry was performed, and a tridecapeptide QQFLPEGQSQSQK, detected in the digest, was chemosynthesized. Oral administration of the tridecapeptide exhibited antidepressant-like effects at a low dose comparable to classical antidepressant in the TST. This also exhibited anti-depressant-like effect in the forced swim test. We named it rice endosperm-derived antidepressant-like peptide (REAP). Intriguingly, intraperitoneal administration had no effect. Orally administered REAP(8-13) but not REAP(1-7) exhibited antidepressant-like activity, suggesting that the C-terminal structure is important for the antidepressant-like effect. We confirmed the presence of REAP, corresponding to rice glutelin type B4(130-142) and B5(130-142), in the digest. The effects of REAP were blocked by both dopamine D1 and D2 antagonists. These results suggest that it exerts its antidepressant-like activity through activation of the dopamine system. Taken together, oral administration of a novel tridecapeptide exhibited antidepressant-like effects via the dopamine system. This is the first report of a rice-derived peptide that exhibits antidepressant-like effects.

The Effect of the Rice Endosperm Protein Hydrolysate on the Subjective Negative Mood Status in Healthy Humans: A Randomized, Double-Blind, and Placebo-Controlled Clinical Trial.

The rice endosperm protein (REP) hydrolysate containing the following rice endosperm protein derived oligopeptides QQFLPEGQSQSQK, LPEGQSQSQK, and pEQFLPEGQSQSQK (a N-terminal pyroglutamate residue-modified peptide) reportedly showed an antidepressant-like effect in an animal model. We investigated the effect of the REP hydrolysate on healthy humans who self-reported mental fatigue with subjectively low vigor. Seventy-six participants (age: 20-64 years) were randomly allocated to two groups. The influence of the REP hydrolysate on the mood state was evaluated in two studies: single intake (Study 1) and repeated intake over 4 weeks (Study 2). A salivary stress marker, Chromogranin A (CgA), was measured in Study 1. The single intake of the REP hydrolysate significantly improved the Profile of Mood Status 2nd edition for adults (POMS 2) subscale of Tension-Anxiety. Additionally, the salivary CgA concentrations were remarkably reduced after the single intake of the REP hydrolysate. Though a single intake of the REP hydrolysate did not significantly influence the other subscales and the TMD of the POMS 2 and the Euthymia Scale, both the subjective and objective results supported the possible effect of the REP hydrolysate on reducing anxiety and nervousness. No significant positive effects on the subjective mood state (Euthymia Scale and POMS 2) and sleep quality (Insomnia Severity Index) were observed in the trial setting employed for Study 2. In conclusion, a single intake of REP hydrolysate might help relax the subjective feelings of tension and anxiety. The effectiveness of repeated REP hydrolysate intake needs to be tested in a different clinical setting.

Rice Endoplasmic Protein-Derived Peptides, Rice-Ghretropins A and B, Stimulate Ghrelin Release in MGN3-1 Cells and Increase Plasma Acylated Ghrelin and Food Intake in Mice.

In this study, we demonstrated that novel rice-derived bioactive peptides promote the secretion of ghrelin, an endogenous orexigenic hormone secreted from the stomach. The enzymatic digest of rice endosperm protein with subtilisin, a microorganism-derived enzyme, stimulated acylated ghrelin secretion in the ghrelin-releasing cell line MGN3-1 and increased food intake after oral administration in mice. By performing a comprehensive analysis based on structure-activity relationships, we selected candidate peptides from over 30,000 peptides in the rice digest. Among them, we found that QAFEPIRSV and TNPWHSPRQGSF, corresponding to the amino acid sequence of the rice endoplasmic proteins glutelin A1 or A2(52-60) and B1 or B2(31-42), respectively, stimulated acylated ghrelin release in MGN3-1 cells. We named them rice-ghretropins A and B. Pyroglutamate formation of rice-ghretropin A, [pyr1]-rice-ghretropin A, also promoted ghrelin secretion. Furthermore, oral administration of rice-ghretropins increased food intake, plasma ghrelin concentration, and small intestinal transit in mice. In addition, the subtilisin digest of the rice protein significantly increased food intake for 4 h in 9 month-old (control: 0.61 ± 0.049 g; digest: 0.83 ± 0.059 g) and 24 month-old mice (control: 0.52 ± 0.067 g; digest: 1.01 ± 0.064 g). In summary, we found that novel bioactive peptides, namely, rice-ghretropins, from the enzymatic digest of rice endosperm stimulated acylated ghrelin secretion and increased food intake. This is the first report of rice-derived exogenous bioactive peptides that increase acylated ghrelin secretion.

Islet1 selectively promotes peripheral axon outgrowth in Rohon-Beard primary sensory neurons.

We isolated a novel zebrafish mutant, lullaby (llb), and showed that the llb locus encodes the zebrafish orthologue of isl1. Rohon-Beard (RB) primary sensory neurons are multipolar neurons that extend their central axons longitudinally within the spinal cord and also extend their peripheral axons under the skin. In llb embryos, the outgrowth of the peripheral axons of RB neurons was selectively impaired, which correlated with down-regulation of the expression of dihydropyrimidinase-like 3 (dpysl3, also known as collapsin response mediator protein 4, crmp4). Antisense morpholino oligonucleotide (AMO)-mediated knockdown of dpysl3 inhibited the outgrowth of the peripheral axons of RB neurons, and semaphorin 3d (sema3d) AMO enhanced this effect. These data indicate that Dpysl3 is cooperating with Sema3d in the peripheral axon outgrowth, and Isl1 is required for the selective outgrowth of the peripheral axons of RB neurons by maintaining the expression of dpysl3.

The effects of bathing in neutral bicarbonate ion water.

Percutaneously absorbed carbon dioxide enhances blood flow. The mechanism by which it does so is unclear, but we hypothesized that it involves bicarbonate ions. BALB/c mice were bathed in neutral bicarbonate ionized water (NBIW) and showed increased blood bicarbonate levels and blood flow via phosphorylation of peripheral vascular endothelial nitric oxide synthase (eNOS) and production of nitric oxide (NO). Phosphorylation of eNOS and NO production were also increased in human umbilical vein endothelial cells cultured in medium containing NBIW, and NBIW showed reactive oxygen species scavenging activity. In a double-blind, randomized study in men and women aged 30 to 59 years with subjective cold intolerance, bathing in NBIW elevated body temperature faster than bathing in a control solution and improved chills and sleep quality. Taken together, our results show that percutaneously absorbed carbon dioxide changes to bicarbonate ions, which act directly on endothelial cells to increase NO production by phosphorylation of eNOS and thus improve blood flow.

Effect of gummy candy containing ubiquinol on secretion of saliva: A randomized, double-blind, placebo-controlled parallel-group comparative study and an in vitro study.

A randomized, double-blind, placebo-controlled, parallel-group comparative clinical study was conducted to examine the effects of ubiquinol (the reduced form of Coenzyme Q10) on secretion of saliva. This interventional study enrolled 40 subjects aged 65 years or younger who were healthy, but noted slight dryness of the mouth. Subjects were randomized with stratification according to gender and age to ingestion of gummy candy containing 50 mg of ubiquinol or placebo twice daily for 8 weeks. At the end of study, along with a significant increase of the CoQ10 level in saliva (p = 0.025*, d = 0.65), there was a significant increase of the saliva flow rate (p = 0.048*, d = 0.66) in the ubiquinol candy group (n = 18; 47.4±6.2 years; 6 men and 12 women) compared to the placebo group (n = 20; 52.2±7.7 years; 4 men and 16 women). The strength of the stomatognathic muscles was not significantly enhanced by ingestion of ubiquinol candy. Compared with baseline, significant improvement of the following four questionnaire items was observed in the ubiquinol group at the end of the study: feeling tired (p = 0.00506, d = -0.726), dryness of the mouth (p = 0.04799, d = -0.648), prone to catching a cold (p = 0.00577, d = -0.963), and diarrhea (p = 0.0166, d = -0.855). There were no serious adverse events. An in vitro study revealed that ubiquinol stimulated a significant and concentration-dependent increase of ATP production by a cell line derived from human salivary gland epithelial cells (p<0.05), while 1 nM ubiquinol significantly suppressed (p = 0.028) generation of malondialdehyde by cells exposed to FeSO4-induced oxidative stress. These findings suggest that ubiquinol increases secretion of saliva by suppressing oxidative stress in the salivary glands and by promoting ATP production. Trial Registration: UMIN-CTR UMIN000024406.

Formation and carbon monoxide-dependent dissociation of Allochromatium vinosum cytochrome c' oligomers using domain-swapped dimers.

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c' from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.