Effects of stress contagion on anxiogenic- and orofacial inflammatory pain-like behaviors with brain activation in mice.

The conditions of stress contagion are induced in bystanders without direct experiences of stressful events. This study determined the effects of stress contagion on masseter muscle nociception in mice. Stress contagion was developed in the bystanders after cohabitating with a conspecific mouse subjected to social defeat stress for 10 days. On Day 11, stress contagion increased anxiety- and orofacial inflammatory pain-like behaviors. The c-Fos and FosB immunoreactivities evoked by masseter muscle stimulation were increased in the upper cervical spinal cord, while c-Fos expressions were increased in the rostral ventromedial medulla, including the lateral paragigantocellular reticular nucleus and nucleus raphe magnus in stress contagion mice. The level of serotonin in the rostral ventromedial medulla was increased under stress contagion, while the number of serotonin positive cells was increased in the lateral paragigantocellular reticular nucleus. Stress contagion increased c-Fos and FosB expressions in the anterior cingulate cortex and insular cortex, both of which were positively correlated with orofacial inflammatory pain-like behaviors. The level of brain-derived neurotrophic factor was increased in the insular cortex under stress contagion. These results indicate that stress contagion can cause neural changes in the brain, resulting in increased masseter muscle nociception, as seen in social defeat stress mice.

Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress.

Rab GTPases, the largest group of small monomeric GTPases, have been shown to participate in membrane trafficking involving many cellular processes. However, their roles during osteoblastic differentiation remain to be elucidated. In this study, we investigated Rab GTPase involvement in osteoblastic differentiation. Protein levels of a series of Rabs (Rab4, Rab5, Rab7, Rab9a, Rab11a/b, and Rab27) were increased during osteoblastic differentiation of MC3T3-E1 cells, and the Rab11a/b levels were particularly pronounced in the presence of Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, an activator of osteoblastogenesis. We subsequently investigated the functional contribution of Rab11a and Rab11b during osteoblastic differentiation. The alkaline phosphatase (ALP) levels were reduced by Rab11b depletion but not by Rab11a depletion. Because our result suggested that Rab11a and Rab11b could be regulated downstream of Runx2 (Runt-related transcription factor 2), a key transcription factor for osteoblastic differentiation, we investigated the effects of the double knockdown of Runx2 and Rab11a or Rab11b on osteoblastic phenotypes. The double knockdown significantly reduced ALP activity as well as collagen deposition compared with single Runx2 knockdown. Furthermore, the Rab11a and Rab11b response to mechanical stress in vivo was investigated using a mouse orthodontic tooth movement model. Rab11a and Rab11b expression was enhanced in the periodontal ligament, where bone formation is activated by tensile stress. This study shows that Rab11a and Rab11b are regulated downstream of Runx2 in osteoblastic differentiation, and their expressions are also controlled by tensile stress.

Structures of the cGMP-dependent protein kinase in malaria parasites reveal a unique structural relay mechanism for activation.

The cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) was identified >25 y ago; however, efforts to obtain a structure of the entire PKG enzyme or catalytic domain from any species have failed. In malaria parasites, cooperative activation of PKG triggers crucial developmental transitions throughout the complex life cycle. We have determined the cGMP-free crystallographic structures of PKG from Plasmodium falciparum and Plasmodium vivax, revealing how key structural components, including an N-terminal autoinhibitory segment (AIS), four predicted cyclic nucleotide-binding domains (CNBs), and a kinase domain (KD), are arranged when the enzyme is inactive. The four CNBs and the KD are in a pentagonal configuration, with the AIS docked in the substrate site of the KD in a swapped-domain dimeric arrangement. We show that although the protein is predominantly a monomer (the dimer is unlikely to be representative of the physiological form), the binding of the AIS is necessary to keep Plasmodium PKG inactive. A major feature is a helix serving the dual role of the N-terminal helix of the KD as well as the capping helix of the neighboring CNB. A network of connecting helices between neighboring CNBs contributes to maintaining the kinase in its inactive conformation. We propose a scheme in which cooperative binding of cGMP, beginning at the CNB closest to the KD, transmits conformational changes around the pentagonal molecule in a structural relay mechanism, enabling PKG to orchestrate rapid, highly regulated developmental switches in response to dynamic modulation of cGMP levels in the parasite.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

Identification and characterization of R2TP in the development of oral squamous cell carcinoma.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells.

HEATR1, a novel interactor of Pontin/Reptin, stabilizes Pontin/Reptin and promotes cell proliferation of oral squamous cell carcinoma.

Pontin and Reptin are closely related proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They form a hetero-oligomeric complex, Pontin/Reptin, which is involved in protein stability and assembly of the protein complexes as a molecular chaperone. Overexpression of Pontin and Reptin in tumor cells has been reported and is implicated in the development of various cancers. However, the molecular mechanism of Pontin/Reptin function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we identify HEAT repeat-containing protein 1 (HEATR1) as a novel binding factor of Pontin/Reptin. Functionally, HEATR1 stabilizes Pontin/Reptin and positively regulates OSCC cell proliferation by activating mTOR and pre-rRNA synthesis. We also find that HEATR1 expression is markedly upregulated in tumor region of OSCC tissue. Hence, we propose that HEATR1 is involved in the regulation of mTOR and ribosome biogenesis as a potential protein stabilizer of Pontin/Reptin in OSCC.

ROCK inhibitors enhance bone healing by promoting osteoclastic and osteoblastic differentiation.

Osteoclast and osteoblast are essential for proper bone development and remodeling as well as recovery of bone fracture. In this study, we seek chemical compounds that enhance turnover of bone metabolism for promoting bone healing. First, we screen a chemical library which includes 378 compounds by using murine pre-osteoclastic RAW264.7 cells to identify compounds that promote osteoclastic differentiation. We find that two ROCK (Rho-associated coiled-coil kinase) inhibitors, HA-1077 (Fasudil) and Y-27632, enhance osteoclastogenesis. Subsequently, we identify that these two compounds also increase osteoblastic differentiation of MC3T3-E1 cells. Finally, our in vivo experiment shows that the local administration of ROCK inhibitors accelerate the bone healing of the rat calvarial defect.

Daily administration of Sake Lees (Sake Kasu) reduced psychophysical stress-induced hyperalgesia and Fos responses in the lumbar spinal dorsal horn evoked by noxious stimulation to the hindpaw in the rats.

We tested whether Sake Lees (SL) had inhibitory effects on hyperalgesia in the hindpaw under psychophysical stress conditions. Male rats were subjected to repeated forced swim stress treatments (FST) from Day -3 to Day -1. Intraperiotoneal administration of SL which contained low concentration of ethanol (SLX) was conducted after each FST. On Day 0, formalin-evoked licking behaviors and Fos responses in the lumbar spinal cord (DH) and several areas within the rostral ventromedial medulla (RVM) were quantified as nociceptive responses. FST-induced hyperalgesia in the hindpaw was prevented by repeated SL and SLX treatments. Fos expression was significantly increased in DH and some areas within the RVM under FST, which was prevented by repeated SL or SLX. These findings indicated that daily administration of SL had the potential to alleviate stress-induced hyperalgesia.

Japanese Rice Wine can reduce psychophysical stress-induced depression-like behaviors and Fos expression in the trigeminal subnucleus caudalis evoked by masseter muscle injury in the rats.

We determined if Japanese Rice Wine (Sake) had inhibitory effects on stress-induced enhancement of masseter muscle (MM) nociception in the rats. Male rats were subjected to the repeated forced swim stress (FS) or sham conditionings from Day -3 to -1. Daily administration of Sake or saline was conducted after each stress conditioning. At Day 0 the number of Fos positive cells, a marker for neural activity, was quantified at the trigeminal subnucleus caudalis (Vc) region by MM injury with formalin. FS increased MM-evoked Fos expression in the Vc region, which was inhibited by Sake compared to saline administration. Sake did not alter the number of Fos positive cells under sham conditions, indicating that inhibitory roles of Sake on neural activity in the Vc region were seen under FS conditions. These findings indicated that Sake had inhibitory roles on stress-induced MM nociception at the Vc region in our experimental conditions.

Analysis of vitamin D receptor binding affinities of enzymatically synthesized triterpenes including ambrein and unnatural onoceroids.

Onoceroids are a rare family of triterpenes. One representative onoceroid is ambrein, which is the main component of ambergris used as a traditional medicine. We have previously identified the onoceroid synthase, BmeTC, in Bacillus megaterium and succeeded in creating ambrein synthase by introducing mutations into BmeTC. Owing to the structural similarity of ambrein to vitamin D, a molecule with diverse biological activities, we hypothesized that some of the activities of ambergris may be induced by the binding of ambrein to the vitamin D receptor (VDR). We demonstrated the VDR binding ability of ambrein. By comparing the structure-activity relationships of triterpenes with both the VDR affinity and osteoclastic differentiation-promoting activity, we observed that the activity of ambrein was not induced via the VDR. Therefore, some of the activities of ambergris, but not all, can be attributed to its VDR interaction. Additionally, six unnatural onoceroids were synthesized using the BmeTC reactions, and these compounds exhibited higher VDR affinity than that of ambrein. Enzymatic syntheses of onoceroid libraries will be valuable in creating a variety of bioactive compounds beyond ambergris.

Multiomics analysis of cultured mouse periodontal ligament cell-derived extracellular matrix.

A comprehensive understanding of the extracellular matrix (ECM) is essential for developing biomimetic ECM scaffolds for tissue regeneration. As the periodontal ligament cell (PDLC)-derived ECM has shown potential for periodontal tissue regeneration, it is vital to gain a deeper understanding of its comprehensive profile. Although the PDLC-derived ECM exhibits extracellular environment similar to that of periodontal ligament (PDL) tissue, details of its molecular composition are lacking. Thus, using a multiomics approach, we systematically analyzed cultured mouse PDLC-derived ECM and compared it to mouse PDL tissue as a reference. Proteomic analysis revealed that, compared to PDL tissue, the cultured PDLC-derived ECM had a lower proportion of fibrillar collagens with increased levels of glycoprotein, corresponding to an immature ECM status. The gene expression signature was maintained in cultured PDLCs and was similar to that in cells from PDL tissues, with additional characteristics representative of naturally occurring progenitor cells. A combination of proteomic and transcriptomic analyses revealed that the cultured mouse PDLC-derived ECM has multiple advantages in tissue regeneration, providing an extracellular environment that closely mimics the environment in the native PDL tissue. These findings provide valuable insights for understanding PDLC-derived ECM and should contribute to the development of biomimetic ECM scaffolds for reliable periodontal tissue regeneration.

The R2TP complex: discovery and functions.

The two closely related AAA+family ATPases Rvb1 and Rvb2 are part of several critical multiprotein complexes, and, thus, are involved in a wide range of cellular processes including chromatin remodelling, telomerase assembly, and snoRNP biogenesis. It was found that Rvb1 and Rvb2 form a tight functional complex with Pih1 (Protein interacting with Hsp90) and Tah1 (TPR-containing protein associated with Hsp90), which are two Hsp90 interactors. We named the complex R2TP. The complex was originally isolated from Saccharomyces cerevisiae and was, subsequently, identified in mammalian cells. R2TP was found to be required for box C/D snoRNP biogenesis in yeast and mammalian cells. More recently, several studies revealed that the complex is also involved in multiple biological processes including apoptosis, phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling, and RNA polymerase II assembly. In this review, we describe the discovery of the complex and discuss the emerging critical roles that R2TP plays in distinct cellular processes.

Preventive Roles of Rice-koji Extracts and Ergothioneine on Anxiety- and Pain-like Responses under Psychophysical Stress Conditions in Male Mice.

This study determined the effect of daily administration of Rice-koji on anxiety and nociception in mice subjected to repeated forced swim stress (FST). In a parallel experiment, it was determined whether ergothioneine (EGT) contained in Rice-koji displayed similar effects. Anxiety and nociception were assessed behaviorally using multiple procedures. c-Fos and FosB immunoreactivities were quantified to assess the effect of both treatments on neural responses in the paraventricular nucleus of the hypothalamus (PVN), nucleus raphe magnus (NRM), and lumbar spinal dorsal horn (DH). FST increased anxiety- and pain-like behaviors in the hindpaw. Rice-koji or EGT significantly prevented these behaviors after FST. In the absence of formalin, both treatments prevented decreased FosB expressions in the PVN after FST, while no effect was seen in the NRM and DH. In the presence of formalin, both treatments prevented changes in c-Fos and FosB expressions in all areas in FST mice. Further, in vitro experiments using SH-SY5Y cells were conducted. Rice-koji and EGT did not affect cell viability but changed the level of brain-derived neurotrophic factor. In conclusion, Rice-koji could reduce anxiety and pain associated with psychophysical stress, possibly mediated by the modulatory effects of EGT on neural functions in the brain.

Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress.

The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

Preclinical models of deep craniofacial nociception and temporomandibular disorder pain.

Chronic pain in temporomandibular disorder (TMD) is a common health problem. Cumulating evidence indicates that the etiology of TMD pain is complex with multifactorial experience that could hamper the developments of treatments. Preclinical research is a resource to understand the mechanism for TMD pain, whereas limitations are present as a disease-specific model. It is difficult to incorporate multiple risk factors associated with the etiology that could increase pain responses into a single animal. This article introduces several rodent models which are often employed in the preclinical studies and discusses their validities for TMD pain after the elucidations of the neural mechanisms based on the clinical reports. First, rodent models were classified into two groups with or without inflammation in the deep craniofacial tissues. Next, the characteristics of each model and the procedures to identify deep craniofacial pain were discussed. Emphasis was directed on the findings of the effects of chronic psychological stress, a major risk factor for chronic pain, on the deep craniofacial nociception. Preclinical models have provided clinically relevant information, which could contribute to better understand the basis for TMD pain, while efforts are still required to bridge the gap between animal and human studies.

Rvb1-Rvb2: essential ATP-dependent helicases for critical complexes.

Rvb1 and Rvb2 are highly conserved, essential AAA+ helicases found in a wide range of eukaryotes. The versatility of these helicases and their central role in the biology of the cell is evident from their involvement in a wide array of critical cellular complexes. Rvb1 and Rvb2 are components of the chromatin-remodeling complexes INO80, Swr-C, and BAF. They are also members of the histone acetyltransferase Tip60 complex, and the recently identified R2TP complex present in Saccharomyces cerevisiae and Homo sapiens; a complex that is involved in small nucleolar ribonucleoprotein (snoRNP) assembly. Furthermore, in humans, Rvb1 and Rvb2 have been identified in the URI prefoldin-like complex. In Drosophila, the Polycomb Repressive complex 1 contains Rvb2, but not Rvb1, and the Brahma complex contains Rvb1 and not Rvb2. Both of these complexes are involved in the regulation of growth and development genes in Drosophila. Rvbs are therefore crucial factors in various cellular processes. Their importance in chromatin remodeling, transcription regulation, DNA damage repair, telomerase assembly, mitotic spindle formation, and snoRNP biogenesis is discussed in this review.

An atlas of chaperone-protein interactions in Saccharomyces cerevisiae: implications to protein folding pathways in the cell.

Molecular chaperones are known to be involved in many cellular functions, however, a detailed and comprehensive overview of the interactions between chaperones and their cofactors and substrates is still absent. Systematic analysis of physical TAP-tag based protein-protein interactions of all known 63 chaperones in Saccharomyces cerevisiae has been carried out. These chaperones include seven small heat-shock proteins, three members of the AAA+ family, eight members of the CCT/TRiC complex, six members of the prefoldin/GimC complex, 22 Hsp40s, 1 Hsp60, 14 Hsp70s, and 2 Hsp90s. Our analysis provides a clear distinction between chaperones that are functionally promiscuous and chaperones that are functionally specific. We found that a given protein can interact with up to 25 different chaperones during its lifetime in the cell. The number of interacting chaperones was found to increase with the average number of hydrophobic stretches of length between one and five in a given protein. Importantly, cellular hot spots of chaperone interactions are elucidated. Our data suggest the presence of endogenous multicomponent chaperone modules in the cell.

R2TP/PAQosome as a promising chemotherapeutic target in cancer.

R2TP/PAQosome (particle for arrangement of quaternary structure) is a novel multisubunit chaperone specialized in the assembly/maturation of protein complexes that are involved in essential cellular processes such as PIKKs (phosphatidylinositol 3-kinase-like kinases) signaling, snoRNP (small nucleolar ribonucleoprotein) biogenesis, and RNAP II (RNA polymerase II) complex formation. In this review article, we describe the current understanding of R2TP/PAQosome functions and characteristics as well as how the chaperone complex is involved in oncogenesis, highlighting DNA damage response, mTOR (mammalian target of rapamycin) pathway as well as snoRNP biogenesis. Also, we discuss its possible involvement in HNSCC (head and neck squamous cell carcinoma) including OSCC (oral squamous cell carcinoma). Finally, we provide an overview of current anti-cancer drug development efforts targeting R2TP/PAQosome.

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein.

Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, "ambrein synthase," for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8-15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid ß-induced apoptosis, were discovered using the synthesized ambrein.