Noboru Ohata

Mycoplasmal Lipoproteins Induce Toll‐Like Receptor 2‐ and Caspases‐Mediated Cell Death in Lymphocytes and Monocytes

Lipoproteins of Mycoplasma salivarium and Mycoplasma fermentans preferentially induced necrotic cell death in lymphocytic cell lines, MOLT‐4 and Raji, and in one monocytic cell line, THP‐1, whereas they preferentially induced apoptotic cell death in another monocytic cell line, HL‐60. These findings were also supported by ultrastructural observations by the use of scanning and transmission electron microscopes and by agarose gel electrophoresis of the genomic DNA. The lipoproteins activated caspase‐3 in both MOLT‐4 and HL‐60 cells, which was assessed by the cleavage of the synthetic substrate DEVD‐pNA and the endogenous substrate poly(ADP‐ribose) polymerase. The cytotoxicity to MOLT‐4 and HL‐60 cells was inhibited by various caspase inhibitors, Ac‐DMQD‐CHO, Ac‐IETD‐CHO, and Z‐VAD‐FMK. The cytotoxicity was also partially suppressed by the monoclonal antibody to Toll‐like receptor 2. Thus this study demonstrated that mycoplasmal lipoproteins induce caspases‐dependent necrotic and apoptotic cell death in lymphocytes and monocytes/macrophages, which is partially induced by TLR2‐mediated signaling.

Signaling Pathways Induced by Lipoproteins Derived from Mycoplasma salivarium and a Synthetic Lipopeptide (FSL-1) in Normal Human Gingival Fibroblasts

Lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL‐1) activate human gingival fibroblasts to induce IL‐6 production and ICAM‐1 expression. Human gingival fibroblasts were treated with lipoproteins or FSL‐1 and then examined for the activation of mitogen‐activated protein kinases (MAPKs), ERK1/2, p38, and SAPK/JNK, and transcription factors, AP‐1 and NF‐κB. Western blotting indicated that p38 and SAPK/JNK were activated in response to the stimulators, but the activation of ERK1/2 could not be discriminated because ERK1/2 was activated in the absence of stimulators. The p38 inhibitor SB 203580 also suppressed their IL‐6 production‐inducing activities, whereas the ERK1/2‐activating MAPK kinase (MEK1) inhibitor PD 98059 did not suppress their activities. Moreover, they were capable of inducing the activation of AP‐1 and NF‐κB. NF‐κB activation was also confirmed by the phosphorylation of IκB‐α. On the basis of these results, it was concluded that lipoproteins of M. salivarium and FSL‐1 are capable of activating the MAPKs p38 and SAPK/JNK and the transcriptional factors AP‐1 and NF‐κB in human gingival fibroblasts.

Craniofacial Pain and Jaw-muscle Activity during Sleep

This study compared the jaw-muscle electromyographic (EMG) activity during sleep in patients with craniofacial pain (n = 63) or no painful conditions (n = 52) and between patients with tension-type headache (TTH: n = 30) and healthy control individuals (n = 30). All participants used a portable single-channel EMG device (Medotech A/S) for four nights. There was no significant difference in EMG activity between craniofacial pain (24.5 ± 17.9 events/hr) and no painful conditions (19.7 ± 14.5), or between TTH (20.8 ± 15.0) and healthy control individuals (15.2 ± 11.6, p >.050). There were positive correlations between EMG activity and number of painful muscles (r = 0.188; p = 0.044), characteristic pain intensity (r = 0.187; p = 0.046), McGill Pain Questionnaire (r = 0.251; p = 0.008), and depression scores (r = 0.291; p = 0.002). Patients with painful conditions had significantly higher night-to-night variability compared with pain-free individuals (p < 0.050). This short-term observational study suggests that there are no major differences between patients with different craniofacial pain conditions and pain-free individuals in terms of jaw-muscle EMG activity recorded with a single-channel EMG device during sleep. However, some associations may exist between the level of EMG activity and various parameters of craniofacial pain. Longitudinal studies are warranted to further explore the relationship between sleep bruxism and craniofacial pain.

Sclerostin Enhances Adipocyte Differentiation in 3T3-L1 Cells.

Sclerostin, a secreted protein encoded by the Sost gene, is produced by osteocytes and is inhibited by osteoblast differentiation and bone formation. Recently, a functional association between bone and fat tissue has been suggested, and a correlation between circulating sclerostin levels and lipid metabolism has been reported in humans. However, the effects of sclerostin on adipogenesis remain unexplored. In the present study, we examined the role of sclerostin in regulating adipocyte differentiation using 3T3‐L1 preadipocytes. In these cells, sclerostin enhanced adipocyte‐specific gene expression and the accumulation of lipid deposits. Sclerostin also upregulated CCAAT/enhancer binding protein β expression but not cell proliferation and caspase‐3/7 activities. Sclerostin also attenuated canonical Wnt3a‐inhibited adipocyte differentiation. Recently, the transcriptional modulator TAZ has been involved in the canonical Wnt signaling pathway. Sclerostin reduced TAZ‐responsive transcriptional activity and TAZ‐responsive gene expression. Transfection of 3T3‐L1 cells with TAZ siRNA increased the lipid deposits and adipogenic gene expression. These results show that sclerostin upregulates adipocyte differentiation in 3T3‐L1 cells, suggesting a possible role for the osteocyte‐derived sclerostin as a regulator of fat metabolism and as a reciprocal regulator of bone and adipose tissues metabolism. J. Cell. Biochem. 117: 1419–1428, 2016.

Analyses of muscular activity, energy metabolism, and muscle fiber type composition in a patient with bilateral masseteric hypertrophy.

ABSTRACT Hyperwork of the masseter muscles due to habitual parafunction is thought to induce masseteric hypertrophy (so called work hypertrophy). However, the causes underlying this disease are not yet fully understood. Recently, we had a patient with bilateral masseteric hypertrophy, and we performed a partial excision of the masseter muscles. In this patients case, we examined muscular activity, energy metabolism, and fiber type composition of the masseter muscles using electromyograms (EMG), 31P-magnetic resonance spectroscopy (MRS), and enzyme-histochemistry. The EMG showed no hyperactivity, and the 31P-MRS showed normal energy spectral patterns and PCr contents of the masseter muscles. The fiber type composition, however, in the muscles in this case was very different from that in muscles with “work hypertrophy” and also that in normal masseter muscles: 1. Loss of type MB fibers; 2. Increases in type IIA and in type IM & IIC fibers; and 3. Decrease in type I fibers. The findings suggest that this is not a case of work hypertrophy but a case of compensatory hypertrophy possibly due to a lack of high-tetanus-tension type IIB fibers.

Symptoms and physiological responses to prolonged, repeated, low-level tooth clenching in humans.

The traditional view contends bruxism, such as tooth grinding/clenching, is part of the etiology of temporomandibular disorders (TMD) including some subtypes of headaches. The purpose of this study is to investigate if a low‐level but long‐lasting tooth‐clenching task initiates TMD symptoms/signs.

A Paced Auditory Serial Addition Task evokes stress and differential effects on masseter-muscle activity and haemodynamics.

This study aimed to determine autonomic and jaw-muscle activities, and haemodynamic responses, to acute experimental mental stress in humans. Eleven healthy men (25.2 ± 3.0 years of age) and five healthy women (23.0 ± 3.7 years of age) performed a standardized mental stress task, the Paced Auditory Serial Addition Task (PASAT). Autonomic function, such as heart rate variability (HRV), and haemodynamic changes were recorded simultaneously. The success rate of the PASAT decreased with increased pace and duration. Low-frequency (5.8 ± 1.1 ms(2)) and high-frequency (5.3 ± 0.6 ms(2)) components of HRV decreased during the PASAT (to 5.0 ± 0.9 ms(2) and 4.6 ± 1.1 ms(2), respectively) as an indication of acute stress. Oxygenated haemoglobin in the masseter muscle (14.6 ± 2.2 10(4) units mm(-3)) remained at an elevated level during the PASAT (15.5 ± 2.5 10(4) units mm(-3)), whereas deoxygenated haemoglobin (7.8 ± 2.3 10(4) units mm(-3)) showed a consistent decrease (to 6.8 ± 2.1 10(4) units mm(-3)). Total haemoglobin and jaw-muscle electromyographic (EMG) activity did not change during the PASAT. Thus, PASAT-induced mental stress changed the parasympathetic/sympathetic balance of the heart and had an acute influence on jaw-muscle haemodynamics, but not on jaw-muscle EMG activity. This non-invasive experimental set-up can be applied to study the effects of repeated or longer-lasting mental stress in order to further the understanding of pathophysiological mechanisms in craniofacial pain conditions.

Attachment Formation After Transplantation of Teeth Cultured With Enamel Matrix Derivative in Dogs

BACKGROUND Implantation of cultured cells may be applied for periodontal regeneration in the future. However, a donor is essential in each case and tooth extraction is required to obtain the periodontal ligament-derived cell. We developed a novel regenerative technique combining tissue culture and transplantation of teeth. The purpose of this study is to evaluate the effect of enamel matrix derivative (EMD) on periodontal healing using this technique in dogs. METHODS A total of 32 incisors from seven beagle dogs were used. The periodontal ligament and cementum 5 mm from the coronal part of the roots were removed, whereas those in the apical part were preserved. Teeth were transplanted after the following treatments: 1) culture with application of EMD to the root surface for 6 weeks (n = 11); 2) culture without application of EMD for 6 weeks (n = 11); and 3) immediately transplanted without culture as control (n = 10). Eight weeks after transplantation, periodontal healing was analyzed. RESULTS The downgrowth of junctional epithelium on the roots of the EMD and culture groups was significantly smaller than that in the control group (P <0.01). Most of the root-planed surfaces in the EMD group were covered with new cementum (72.2% ± 8.6%). This was significantly greater than that in the culture (29.1% ± 22.9%) and control groups (0.3% ± 1.1%). CONCLUSIONS Transplantation of tissue-cultured teeth decreased epithelial downgrowth and increased connective tissue attachment on the root-planed surface. Furthermore, EMD could remarkably increase the new connective tissue attachment in this periodontal regenerative technique.

Diagnostic validity of self-reported measures of sleep bruxism using an ambulatory single-channel EMG device

PURPOSE Self-reported measures have been widely used to indicate the presence of possible and probable sleep bruxism (SB) in both research and clinical situations. However, few studies have attempted to assess the diagnostic validity of this approach. The aim of this study was to estimate the diagnostic validity of self-reported measures of SB using an ambulatory single-channel electromyographic (EMG) device. METHODS A total of 115 participants were enrolled and examined by standardized Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) including two questions related to SB: self-reported SB and morning-jaw symptoms. An ambulatory single-channel EMG device (GrindCare3™, Medotech A/S) was used for measuring jaw-muscle EMG activity during sleep for seven consecutive nights. Cut-off values for different measures of EMG activity (average, maximum and minimum) and the coefficient of variation (CV) were selected to divide participants into two groups, with higher or lower EMG activity or CV values. The sensitivity and specificity for each question and combination of them were calculated. RESULTS Self-reported SB had the highest sensitivity (compared with morning-jaw symptoms) for all measures of EMG activity and CV, although the values were low to modest (average: 76.0%, maximum: 76.9%, minimum: 77.3%, CV: 61.0%). The specificity was low for both the questions related to the different measures of EMG activity and CV (35.1-52.4%). CONCLUSIONS This study indicated that the diagnostic validity of self-reported measures of SB was low to modest using an ambulatory EMG device assessment as a reference. Using only self-reported measures for the assessment of SB may not have a high validity, which should be taken into consideration in the clinical evaluation of patients.

Does restriction of mandibular movements during sleep influence jaw‐muscle activity?

To investigate the effect of restriction of mandibular movements during sleep on jaw-muscle electromyographic (EMG) activity. Eleven healthy subjects (four men and seven women; age, 25·9 ± 3·1 years) with self-reports of sleep bruxism participated in three randomised sessions with three different types of oral appliances: (i) full-arch maxillary and mandibular appliances which did not allow any mandibular movement, that is, restrictive oral appliance (restrict-MMOA), (ii) full-arch maxillary and mandibular oral appliances (free-MMOA) with no restrictions of mandibular movements and (iii) conventional full-arch flat stabilisation appliance, that is, maxillary oral appliance (free-MOA). Baseline recordings (1st EMG recording) of jaw-muscle activity during sleep without any oral appliance were performed and followed by 1 week of nightly use of each oral appliance (three sessions). During the last night in each session, jaw-muscle activity was recorded (2nd, 3rd and 4th EMG recordings) and compared to baseline values. All EMG data were analysed in accordance with the gold-standard diagnostic method. The average jaw-muscle activity expressed as number of EMG episodes and bursts per hour sleep was significantly reduced during any combination of appliance compared to baseline values. The inhibitory effect of the appliances was specific to the number of phasic EMG episodes and bursts (P < 0·01), with no effects on tonic EMG bursts or episodes (P > 0·30). The results indicated that restriction of mandibular movements with oral appliances do not have any major influence on jaw-muscle activity during sleep but rather that the immediate effect of any combination of oral appliances lead to a suppression of phasic EMG bursts and episodes.