Effect of Gum Chewing on PFC Activity During Discomfort Sound Stimulation.

The prefrontal cortex (PFC) is sensitive to the stress exposure and involved in stress coping. And the effects of gum chewing on the stress have been studied using NIRS. However, when measuring NIRS on PFC during gum chewing, blood flows in shallow tissues (scalp, skin, muscle) might be affected. A NIRS used in the present study first, which has a short distance (1 cm) and the usual (3 cm) source-detector (S-D) regression, can allow eliminating shallow tissues effect of gum chewing. The aim of this study was to investigate the hypothesis that gum chewing activates the right prefrontal cortex (PFC) in stress coping against negative sounds (NS) from the International Affective Digitized Sounds-2 (IADS) as a mental stress task. NS showed activation in the right PFC. There was a significant difference between NS, and NS with Gum, where NS with Gum showed an increased PFC activity, increased alpha wave appearance rate, a higher value in heart rate level, and a higher VAS score indicating 'pleasant'. Gum chewing activated right PFC activity while exposed to negative sounds from IADS as a mental stress task.

Role of mycorrhizal associations in tree spatial distribution patterns based on size class in an old-growth forest.

An important factor controlling tree species diversity is conspecific density dependence (CDD). Adult trees associated with arbuscular mycorrhiza (AM) and ectomycorrhiza (ECM) can exhibit negative and positive CDD effects on conspecific recruitment, respectively. However, the extent to which these mycorrhizal associations affect spatial distributions of individual trees and their relative abundances within forests through CDD remains uncertain. We analysed changes in spatial correlations between adults and conspecific juveniles at different growth stages of five hardwood species in a 6-ha plot of an old-growth forest using a point pattern analysis. The clump sizes of large individuals were also evaluated using the Iδ index (a measure of individual dispersion) in 24 species. In two AM-associated species, juveniles were distributed at greater distances with increasing size or were always distributed at a distance from adults, resulting in small clumps of adults. In contrast, juveniles of two ECM-associated species were distributed close to adults during early or late growth stage, resulting in large clumps of adults. Juveniles of an ECM-associated species disappeared with increasing size, probably due to shade intolerance. In 24 tree species with large numbers of individuals within a plot, the relative basal area was related to both mycorrhizal type and maximum diameter, suggesting that the relative abundance of a species is largely related to its mycorrhizal associations and maximum plant size. This study strongly demonstrated that mycorrhizal associations play an important role in determining the spatial distribution patterns and community structure of tree species through CDD.

Relationships Between Gum Chewing and Stroop Test: A Pilot Study.

Cognitive function tends to decrease with aging, therefore maintenance of this function in an aging society is an important issue. The role of chewing in nutrition is important. Although several studies indicate that gum chewing is thought to improve cognitive function, it remains debatable whether gum-chewing does in fact improve cognitive function. The Stroop test is a psychological tool used to measure cognition. A shorter reaction time indicates a mean higher behavioral performance and higher levels of oxy-Hb concentration. fNIRS is a powerful, non-invasive imaging technique offering many advantages, including compact size, no need for specially equipped facilities, and the potential for real-time measurement. The left dorsolateral prefrontal cortex (DLPFC) seems to be mainly involved in the Stroop task.The aim of the present study was to investigate the hypothesis that gum-chewing changes cerebral blood flow in the left DLPFC during the Stroop test, and also changes the reaction time. Fourteen healthy volunteers (mean age 26.9 years) participated in this study after providing written informed consent. A piece of tasteless gum weighing 1.0 g was used. Each session was designed in a block manner, i.e. 4 rests (30 s) and 3 blocks of task (30 s). A computerized Stroop test was used (including both congruent and incongruent Stroop tasks) which calculates a response time automatically. The Binominal test was used for comparisons (p < 0.05). The results show activation of the left DLPFC during the Stroop task and that gum chewing significantly increases responses/oxy-Hb concentration and significantly shortens the reaction time.

Tumour-suppressive function of SIRT4 in human colorectal cancer.

BACKGROUND: SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers. METHODS: We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry. RESULTS: SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis. CONCLUSIONS: SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.

CD10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma.

BACKGROUND: Cancer stem cells (CSCs) are responsible for treatment failure. However, their identification and roles in resistance are not well established in head and neck squamous cell carcinoma (HNSCC). METHODS: Three HNSCC cell lines (FaDu, Detroit562 and BICR6) were treated with cisplatin or radiation. Cell surface antigens were analysed by LyoPlate, a novel cell surface antigen array. The expression levels of antigens highly expressed after treatments were further compared between cisplatin-resistant Detroit562 cells and its parental line. Association of the candidate antigen with CSCs properties, namely sphere formation and in vivo tumourigenicity, was also examined. RESULTS: CD10, CD15s, CD146 and CD282 were upregulated across the treated cell lines, while the increased expression of CD10 was prominent in the cisplatin-resistant cell line. Isolation mediated by FACS revealed that the CD10-positive subpopulation was more refractory to cisplatin, fluorouracil and radiation than the CD10-negative subpopulation. It also showed an increased ability to form spheres in vitro and tumours in vivo. Moreover, the CD10-positive subpopulation expressed the CSC marker OCT3/4 at a higher level than that in the CD10-negative subpopulation. CONCLUSIONS: CD10 is associated with therapeutic resistance and CSC-like properties of HNSCC. CD10 may serve as a target molecule in the treatment of refractory HNSCC.

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer.

BACKGROUND: Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance. METHODS: We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining. RESULTS: The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients. CONCLUSIONS: miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

Potential of polar lipids from bovine milk to regulate the rodent dorsal hair cycle.

Among the lipids in bovine milk, minor components such as conjugated linoleic acids and phospholipids are more attractive than triacylglycerols from the standpoint of biological activity. To explore novel functions of bovine milk polar lipids (MPL), topical application to murine dorsal skin was introduced as an assay system. The acetone-insoluble lipid fraction derived from bovine milk was dispersed in ethanol and applied to 9-wk-old C57BL/6N female mice for 3 wk. In combination with visual assessment of the dorsal pigmentation, the progression of the hair cycle was estimated by calculating the ratio of subcutis to dermis thickness. The administration of MPL led to earlier progression of the hair cycle compared with administration of the vehicle. In some cases, the extent of MPL-induced hair cycle progression was comparable to that in animals treated with minoxidil, the most well-known reagent that initiates anagen. These results indicate that the MPL preparation contains a dermal penetrative component that can regulate the hair cycle and, thus, this preparation possesses potential for cosmetic use.

Enzyme structure with two catalytic sites for double-sieve selection of substrate.

High-fidelity transfers of genetic information in the central dogma can be achieved by a reaction called editing. The crystal structure of an enzyme with editing activity in translation is presented here at 2.5 angstroms resolution. The enzyme, isoleucyl-transfer RNA synthetase, activates not only the cognate substrate L-isoleucine but also the minimally distinct L-valine in the first, aminoacylation step. Then, in a second, "editing" step, the synthetase itself rapidly hydrolyzes only the valylated products. For this two-step substrate selection, a "double-sieve" mechanism has already been proposed. The present crystal structures of the synthetase in complexes with L-isoleucine and L-valine demonstrate that the first sieve is on the aminoacylation domain containing the Rossmann fold, whereas the second, editing sieve exists on a globular beta-barrel domain that protrudes from the aminoacylation domain.

The 2.0 A crystal structure of Thermus thermophilus methionyl-tRNA synthetase reveals two RNA-binding modules.

BACKGROUND: The 20 aminoacyl-tRNA synthetases are divided into two classes, I and II. The 10 class I synthetases are considered to have in common the catalytic domain structure based on the Rossmann fold, which is totally different from the class II catalytic domain structure. The class I synthetases are further divided into three subclasses, a, b and c, according to sequence homology. No conserved structural features for tRNA recognition by class I synthetases have been established. RESULTS: We determined the crystal structure of the class Ia methionyl-tRNA synthetase (MetRS) at 2.0 A resolution, using MetRS from an extreme thermophile, Thermus thermophilus HB8. The T. thermophilus MetRS structure is in full agreement with the biochemical and genetic data from Escherichia coli MetRS. The conserved 'anticodon-binding' residues are spatially clustered on an alpha-helix-bundle domain. The Rossmann-fold and anticodon-binding domains are connected by a beta-alpha-alpha-beta-alpha topology ('SC fold') domain that contains the class I specific KMSKS motif. CONCLUSIONS: The alpha-helix-bundle domain identified in the MetRS structure is the signature of the class Ia enzymes, as it was also identified in the class Ia structures of the isoleucyl- and arginyl-tRNA synthetases. The beta-alpha-alpha-beta-alpha topology domain, which can now be identified in all known structures of the class Ia and Ib synthetases, is likely to dock with the inner side of the L-shaped tRNA, thereby positioning the anticodon stem.

The substrate-binding site in Escherichia coli cyclophilin A preferably recognizes a cis-proline isomer or a highly distorted form of the trans isomer.

The three-dimensional structure of Escherichia coli cytosolic cyclophilin A (CyPA) complexed with a tripeptide (succinyl-Ala-Pro-Ala-p-nitroanilide) was refined at 1.8 A resolution by the multiple isomorphous replacement method to a crystallographic R-factor of 17.6%. As in human CyPA, the peptide binding site in E. coli enzyme is in a cleft created on the surface of the upper sheet of two orthogonal beta-sheets. In this cleft, the walls of the hydrophobic pocket are formed by the side-chains of five non-polar residues, Phe48, Met49, Phe107, Leu108, and Try120, with Phe99 at the bottom. When the cis isomer of the tripeptide binds to the enzyme, a cis-proline ring is inserted into the hydrophobic pocket. Since the binding pocket of CyPAs are largely hydrophobic, the cis isomer of a peptide can be bound more firmly than the trans isomer. Distortion of the trans isomer could lead to better binding, but at an energetic cost of the distortion energy. At the periphery of the upper beta-sheet in E. coli CyPA, conformations of loops L1, L3, and L4 and the segment connecting alpha1 and beta3 with deletions or insertions against human CyPA differ significantly from those in human CyPA. The refined model also shows that steric hindrance to attachment of cyclosporin A (CsA) prevents E. coli CyPA forming a complex with CsA. Thus, the extra amino acid residue of E. coli CyPA, polar Gln89, lies along the pathway to the hydrophobic pocket of CyPA and seems to prevent the access hydrophobic part of CsA to the cleft of CyPA.

Crystal structure of Escherichia coli methionyl-tRNA synthetase highlights species-specific features.

The 3D structure of monomeric C-truncated Escherichia coli methionyl-tRNA synthetase, a class 1 aminoacyl-tRNA synthetase, has been solved at 2.0 A resolution. Remarkably, the polypeptide connecting the two halves of the Rossmann fold exposes two identical knuckles related by a 2-fold axis but with zinc in the distal knuckle only. Examination of available MetRS orthologs reveals four classes according to the number and zinc content of the putative knuckles. Extreme cases are exemplified by the MetRS of eucaryotic or archaeal origin, where two knuckles and two metal ions are expected, and by the mitochondrial enzymes, which are predicted to have one knuckle without metal ion.

Crystal structure of the E166A mutant of extended-spectrum beta-lactamase Toho-1 at 1.8 A resolution.

Bacterial resistance to beta-lactams is mainly due to the production of beta-lactamase. Especially through the production of extended-spectrum beta-lactamases (ESBLs), bacteria have acquired resistance not only to penicillins, but also to expanded-spectrum cephems. Here, we describe the crystal structure of the E166A mutant of class A beta-lactamase Toho-1 at 1.8 A resolution, the first reported tertiary structure of an ESBL. Instead of the wild-type enzyme, a mutant Toho-1, in which Glu166 was replaced with alanine, was used for this study, because of the strong tendency of the wild-type enzyme to form twinned crystals. The overall structure of Toho-1 is similar to the crystal structures of non-ESBLs, with no pronounced backbone rearrangement of the framework. However, there are some notable local changes. First, a difference in the disposition of an arginine residue, which is at position 244 in non-ESBLs but at position 276 in Toho-1 and other ESBLs, was revealed and the role of this arginine residue is discussed. Moreover, changes in the hydrogen-bonding pattern and in the formation of the hydrophobic core were also observed near the Omega loop. In particular, the lack of hydrogen bonds in the vicinity of the Omega loop could be a cause of the extended substrate specificity of Toho-1. Through the generation of a model for the enzyme-substrate complex, a conformational change of Toho-1 occurring on complex formation is discussed based on the active-site cleft structure and the substrate profile.

Interaction of late murine erythroid progenitors and stroma involves a recognition mechanism with fucosyl specificity.

We have previously reported that the specific recognition and binding of murine hemopoietic progenitors spleen colony-forming units (CFU-S) and granulocyte-macrophage CFU (CFU-GM) to hemopoietic stroma is dependent upon a membrane recognition system with galactose and mannose specificities. By using synthetic neoglycoproteins with galactose, mannose, or fucose covalently bound to bovine serum albumin (BSA) in standard long-term bone marrow cultures (LTBMC), galactosyl-BSA (gal-BSA) and mannosyl-BSA (man-BSA) but not fucosyl-BSA (fuc-BSA) inhibited the binding of CFU-S and CFU-GM to the stromal layer. In the present work it was shown that binding of erythroid burst-forming units (BFU-E) to stroma in standard LTBMC is also inhibited by gal-BSA and man-BSA. We then studied a different system of LTBMC that favored erythropoiesis and allowed the production of erythroid CFU (CFU-E) for 4 weeks. In the presence of the fuc-BSA as well as gal-BSA and man-BSA, total cell production and CFU-E production were halted in the supernate as well as the adherent layer. These results indicate the presence of a fucosyl recognition system on the surface of the late erythroid precursors, CFU-E.

Murine spleen culture: homing of hemopoietic progenitor cells to spleen is not mediated by a similar mechanism to that in marrow.

We have previously shown that in long-term bone marrow cultures (LTMC) the specific recognition and binding of hemopoietic stem cells to stroma, which we call "homing," is mediated by a recognition mechanism involving a surface membrane lectin with galactosyl and mannosyl specificities. Subsequent in vivo studies in lethally irradiated mice confirmed that homing to the marrow similarly involves a galactosyl- and mannosyl-specific recognition mechanism. However, these in vivo studies suggested that homing of hemopoietic progenitor cells to spleen was based upon a different molecular recognition mechanism. In the present study splenic homing was investigated in a cell culture system composed of an adherent layer of splenic stromal cells inoculated with stroma-free stem cells from the supernate of LTMC. In this system, splenic stroma supported proliferation and differentiation of hemopoietic precursors for a few weeks. When stem cells were added to the cultures in the presence or absence of inhibitory concentrations of neoglycoprotein reagents specific for galactosyl, mannosyl, or fucosyl lectins, the pattern of production of total cells, pluripotential stem cells (CFU-S), and granulocyte-macrophage committed progenitors (CFU-GM) remained the same. These data support our in vivo observations that homing of stem cells to splenic stroma is not mediated by a surface lectin with galactosyl and mannosyl specificities as it is in bone marrow, but rather by a different molecular mechanism.

Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow.

Stromal cell lines, designated MS-1, -2, -3, -4, -5, -6, and -7 were established by irradiating the adherent cells in long-term bone marrow cultures with 900-rad x-rays. Two of the cell lines, MS-1 and MS-5, have the capacity to support the growth of hemopoietic stem cells (spleen colony-forming cells and granulocyte-macrophage colony-forming cells) for greater than 2 months in vitro. These two cell lines were alkaline phosphatase-, peroxidase-, and factor VIII-negative and positive for periodic acid-Schiff and nonspecific esterase. Extracellular matrix proteins such as fibronectin, laminin, and collagen type I were produced by these two cell lines. Neither MS-1 cell- nor MS-5 cell-conditioned medium supported the growth of hemopoietic stem cells, and hemopoietic stem cells were found preferentially to be under and on MS-1 and MS-5 layers rather than in suspension. Close contact with the MS-1 cell layer or the MS-5 cell layer appears to be essential in maintaining hemopoiesis in vitro. Conditioned media from MS-1 cells and MS-5 cells stimulated granulocyte colony formation from murine bone marrow cells in semisolid culture.

Generation of virtual models for planning orthognathic surgery using a modified multimodal image fusion technique.

Streak artefacts caused by dental metals deteriorate the quality of computed tomography (CT) images. We developed and evaluated a method for generating three-dimensional virtual models to plan orthognathic surgery in patients with multiple dental materials, to avoid the adverse effects of metal artefacts in image fusion. The method basically consists of four procedures: (1) fabrication of a splint in the open-mouth position with fiducial markers, (2) reconstruction of a virtual skull model in the open-mouth position from CT scanning, (3) reconstruction of two virtual dental models in the open-mouth position and either the intercuspal position (ICP) or centric relation (CR) from surface scanning, and (4) three serial steps of image registration and subsequent repositioning of the mandible to the ICP or CR. This method allows for the registration of skull and dental models under artefact-free conditions. To validate the method, CT and dental cast data from 30 patients were used. The registration accuracy was 0.080 mm for the initial registration, 0.033 mm for the second registration, and 0.028 mm for the third registration. The present method can be used to determine the occlusal relationships and craniofacial morphology of patients with dental metals and can be applied to computer-assisted diagnosis and surgery.

New potent antagonists of leukotrienes C4 and D4. 1. Synthesis and structure-activity relationships.

(p-Amylcinnamoyl)anthranilic acid (3a) had moderate antagonist activities against LTD4-induced smooth muscle contraction on guinea pig ileum and LTC4-induced bronchoconstriction in anesthetized guinea pigs. Modifications were made in the hydrophobic part (cinnamoyl moiety) and the hydrophilic part (anthranilate moiety) of 3a. A series of 8-(benzoylamino)-2-tetrazol-5-yl-1,4-benzodioxans and 8-(benzoylamino)-2-tetrazol-5-yl-4-oxo-4H-1-benzopyrans were revealed to be potent antagonists of leukotrienes C4 and D4. Among both series, ONO-RS-347 (18k) and ONO-RS-411 (19h) were the most potent and orally active antagonists, respectively. Structure-activity relationships are discussed.

Synthesis and 5-lipoxygenase inhibitory activities of eicosanoid compounds.

Ten eicosanoid compounds (3, 6, 9, 11, 12, 15, 18, 21, 23, and 25), methyl (6E,8Z,11Z,14Z)-5-hydroxy-6,8,11,14-eicosatetraenoate (5-HETE, 10), leukotriene A4 (26), and (5S,6E,8E,10E,12RS,14E)-5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE, 27) were prepared and their inhibitory activities against the 5-lipoxygenase from guinea pig polymorphonuclear leukocytes (PMNL) were tested. 5,6-Methanoleukotriene A4 (18) was especially a potent and specific inhibitor of the 5-lipoxygenase without inhibiting the cyclooxygenase and the 12-lipoxygenase. Leukotriene A4, 5-HETE, and 5,12-diHETE also have inhibitory activities against the 5-lipoxygenase at micromolar concentrations, which can regulate the formation of slow-reacting substance of anaphylaxis intracellulary.

Allelotype analysis of gallbladder carcinoma associated with anomalous junction of pancreaticobiliary duct.

Anomalous junction of pancreaticobiliary duct (AJPBD) patients has an increased risk of gallbladder and bile duct carcinomas. However, the relevance of carcinoma with AJPBD is not fully clarified. We performed analysis of loss of heterozygosity (LOH) at p53 locus and immunohistochemistry of p53 and K-ras gene mutation in five cases of gallbladder carcinoma associated with AJPBD. LOH of p53 locus and overexpression of p53 were detected in two out of five (40%) and five out of five (100%), respectively, in the present study. K-ras gene mutation at codon 12 and 13 was not detected (0%, 0/5). These results suggest that aberrations of p53 are involved in carcinogenesis of gallbladder carcinoma associated with AJPBD. Next, in order to find the genetic events besides K-ras mutation and overexpression of mutant p53 in this disease, LOH analysis was performed using 72 microsatellite markers. High frequency of allelic loss (> 50%) was found on 2p (81.8%), 4p (50%), 4q (50%), 8q (60%), 9q (50%), 10p (50%), 14p (60%), 14q (50%), 16p (60%), 19p (50%), 21p (50%) and Xp (66.6%). The highest deletion regions on chromosome 2p24 (3/3, 100%), 14q22 (3/4, 75%) and 21q22 (3/4, 75%) were found. The present study suggests that gallbladder carcinoma associated with AJPBD has high frequent allelic loss and has two new regions which may harbor putative tumor suppressor genes.

Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura.

Upshaw-Schulman syndrome (USS) is a congenital bleeding disorder characterized by repeated episodes of thrombocytopenia and microangiopathic hemolytic anemia that respond to infusions of fresh frozen plasma. Inheritance of USS has been thought to be autosomal recessive, because 2 siblings in the same family are often affected but their parents are asymptomatic. Recently, chronic relapsing thrombotic thrombocytopenic purpura (CR-TTP), reported almost exclusively in adults, was shown to be caused by inherited or acquired deficiency in the activity of a plasma von Willebrand factor-cleaving protease (vWF-CPase). The pathogenesis of USS is unknown, and a relationship between CR-YEP and USS has not been reported. We studied 3 unrelated USS patients (ST, SY, and KI) who presented with severe indirect neonatal hyperbilirubinemia. All 3 patients had undetectable vWF-CPase activity, and the inhibitors to vWF-CPase were all negative. In their parents with no clinical symptoms, vWF-CPase activities as a percentage of control samples (mother/father) were 17/20 for ST, 60/45 for SY, and 36/5.6 for KI. Thus, USS and vWF-CPase activity appear to be coinherited as autosomal recessive traits. Transfusion of fresh frozen plasma in 2 patients (ST and SY) resulted in the expected maximal increment of approximately 7% to 8% in vWF-CPase activity at 1 to 4 hours, but the levels became less than 3% within 2 days. After this decrease, platelet counts increased, plateaued in the normal range at 10 to 12 days, and declined thereafter. Thus, the 2 to 3 weeks of therapeutic benefit from plasma infusions will be discussed in relation to the intravascular lifetime of vWF-CPase.