Bone metabolism induced by denture insertion in positron emission tomography.

18F-fluoride positron emission tomography (PET) can identify subtle functional variation prior to the major structural change detectable by X-ray. This study aims to investigate the mechanobiological bone reaction around the abutment tooth and in the residual ridge, induced by insertion of removable partial denture (RPD) within two different groups of patients: patients without denture experience (Group 1) and patients with denture experience before (Group 2), using 18F-fluoride PET imaging technique. 18F-fluoride PET/computerised tomography (CT) scan was performed to examine the bone metabolic change in mandible before and after the RPD treatment. Region of interests (ROIs) were placed in alveolar bone around abutment tooth and in residual bone beneath the RPD. Standardised uptake value (SUV), reflecting the accumulation of 18F-fluoride, was measured for each ROI. In all subjects of Group 1, SUVs after insertion were higher than before in both alveolar bone and residual bone, while there was less significant change in SUV in subjects of Group 2. This study demonstrated using longitudinal 18F-fluoride PET scans to effectively examine the bone metabolic change in mandible induced by occlusal loading after RPD insertion. Using this technique, within the six subjects in this study, it was shown that bone metabolism around abutment tooth and residual ridge increased after RPD insertion in case of first-time denture user, while there was no big change in the patient with experience of denture before. This study revealed the effectiveness of applying PET to evaluate bone metabolic activity as mechanobiological reaction.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium WST-1.

Rapid susceptibility testing for slowly growing nontuberculous mycobacteria (NTM) using a colorimetric microbial viability assay based on the reduction of the water-soluble tetrazolium salt {2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1)} using 2,3,5,6-tetramethyl-1,4-benzoquinone as an electron mediator was developed. Using the Clinical and Laboratory Standards Institute (CLSI) method, a long-term incubation time (7-14 days) was required to determine the minimum inhibitory concentrations (MICs) of the slowly growing NTM. The MICs for a variety of different antibiotics against the slowly growing NTM were determined by the WST-1 colorimetric method and compared with those obtained using the broth microdilution methods approved by the CLSI. Good agreement was found between the MICs determined after 3-4 days using the WST-1 colorimetric method and those obtained after 10-14 days using the broth microdilution method. The results suggest that the WST-1 colorimetric assay is a useful method for the rapid determination of the MICs for the slowly growing NTM.

Mechanobiological bone reaction quantified by positron emission tomography.

While nuclear medicine has been proven clinically effective for examination of the change in bone turnover as a result of stress injury, quantitative correlation between tracer uptake and mechanical stimulation in the human jawbone remains unclear. This study aimed to investigate the relationship between bone metabolism observed by 18F-fluoride positron emission tomography (PET) images and mechanical stimuli obtained by finite element analysis (FEA) in the residual ridge induced by the insertion of a removable partial denture (RPD). An 18F-fluoride PET/CT (computerized tomography) scan was performed to assess the change of bone metabolism in the residual ridge under the denture before and after RPD treatment. Corresponding patient-specific 3D finite element (FE) models were created from CT images. Boundary conditions were prescribed by the modeling of condylar contacts, and muscular forces were derived from the occlusal forces measured in vivo to generate mechanobiological reactions. Different mechanobiological stimuli, e.g., equivalent von Mises stress (VMS), equivalent strain (EQV), and strain energy density (SED), determined from nonlinear FEA, were quantified and compared with the standardized uptake values (SUVs) of PET. Application of increased occlusal force after RPD insertion induced higher mechanical stimuli in the residual bone. Accordingly, SUV increased in the region of residual ridge with higher mechanical stimuli. Thus, with SUV, a clear correlation was observed with VMS and SED in the cancellous bone, especially after RPD insertion (R(2) > 0.8, P < 0.001). This study revealed a good correlation between bone metabolism and mechanical stimuli induced by RPD insertion. From this patient-specific study, it was shown that metabolic change detected by PET in the loaded bone, in a much shorter duration than conventional x-ray assessment, is associated with mechanical stimuli. The nondestructive nature of PET/CT scans and FEA could potentially provide a new method for clinical examination and monitoring of prosthetically driven bone remodeling.

A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay.

UNLABELLED: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on the reduction in a tetrazolium salt 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) with 2-methyl-1,4-napthoquinone as the electron mediator was developed. The proposed method was useful to measure the proliferation of 18 kinds of moulds and seven kinds of yeasts, including representative pathogens such as Aspergillus spp., Candida spp. and Cryptococcus spp. Linear relationships between the absorbance and viable fungal cell density were obtained for all fungi, suggesting that the absorbance change reflected the fungal proliferation. In addition, the minimum inhibitory concentrations (MICs) against a variety of different pathogenic moulds and yeasts for amphotericin B, itraconazole and 5-flucytosine were determined by susceptibility testing using the proposed method and compared with those obtained using the conventional broth microdilution method. There was an excellent agreement between the results obtained using the WST-8 colorimetric method and those obtained using the conventional Clinical and Laboratory Standard Institute method. The WST-8 colorimetric assay is a useful method for rapid determination of accurate MICs for a variety of different fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: A rapid microplate method for the proliferation assay of fungi and the antifungal susceptibility testing using the colorimetric microbial viability assay based on reduction in a tetrazolium salt (WST-8) was developed. The WST-8 colorimetric method was useful to measure the proliferation of a variety of different fungi. In the antifungal susceptibility testing, there was a good agreement between the MICs determined after 24 h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The proposed method was superior to conventional methods in terms of its rapidity towards a variety of different fungi.

Cyclooxygenase-2 activity is important in craniofacial fracture repair.

The aim of this study was to examine the effect of cyclooxygenase (COX)-2 on bone repair after craniofacial fracture in mice. A 4-mm fracture was created in the parietal bone of 8-week-old male COX-2 wild-type (COX-2(+/+)) and knockout (COX-2(-/-)) mice. Ribonucleic acid was extracted from the fractured bone and analysed. For morphological and histological analysis, the mice were killed 8 and 12 weeks after treatment, and sections were prepared. Three-dimensional computed tomography was performed, and the sections were stained with hematoxylin-eosin for histological examination. Expression of COX-2 messenger ribonucleic acid was induced in COX-2(+/+) mice, but not in COX-2(-/-) mice. Ossification at the fracture site was almost complete 12 weeks after fracture in COX-2(+/+) mice. In COX-2(-/-) mice, incomplete union had occurred at the fracture site. In both types of mice, the fracture site contained no cartilaginous tissue, and the callus formed from the periosteal side. These results suggest that COX-2 plays an important role in craniofacial fracture repair and that COX-2-selective non-steroidal anti-inflammatory drugs might interfere with fracture repair of the membranous viscerocranium in the clinical setting.

Anticipation and motor control on repetitive tooth tapping produced by open-close jaw movements.

This study was designed to evaluate the relationship between the temporal anticipation effects and motor control on repetitive tooth tapping movements (tooth tapping produced by open-close jaw movements) commanded by six frequencies of acoustic signals. Ten dentulous young adults were asked to perform tooth tapping movements to acoustic command signals at constant frequencies of 0.3, 0.8, 1.3, 1.8, 2.3 and 2.8 Hz. Surface electromyograms of the Masseter muscles, vertical component of the Mandibular kinesiograph, and acoustic signals were simultaneously recorded. Some parameters were measured for tappings at the last signal (TL) and surplus tappings without signals after the last signal (TL(n)) in each frequency command signal. The results showed that changes in the strategy for tapping movement controls occurred between 0.3 and 0.8 Hz. Between 0.8 and 2.8 Hz, each tooth tapping was automatically initiated before the confirmation of the signal; initiation and control of repetitive tapping movements were apparently based on effector anticipation (predicting the duration of internal processes for a planned movement so that it can be made coincidental to some anticipated external event) and receptor anticipation (anticipation of the arrival of a stimulus due to sensory information about its time of arrival). However, at 0.3 Hz, the movement initiation was delayed, such that each tooth tapping was initiated after the confirmation of the signal. Therefore, tappings at 0.3 Hz showed less dependence on receptor anticipation. These changes in motor control strategy are considered to be a reasonable way to prevent incorrect movements and to minimize erroneous movements.

Anticipatory and reflexive neck muscle activities during voluntary rapid jaw opening and passive jaw depression in humans.

The characteristics of head movement during voluntary rapid jaw opening movement and passive jaw depression were investigated using accelerometers and electromyographs (EMG) on eight healthy examinees. Passive depressions were executed by means of load on the lower jaw, initiated either by examinees themselves or an experimenter. In the depression initiated by examinees, a head-extension movement that preceded the load to the lower jaw and anticipatory activities in the nuchal region of the trapezius muscle were observed. In the depression initiated by the experimenter, the anticipatory activities were not observed. In both of these cases, stretch reflexes were induced in the trapezius muscle. During voluntary rapid jaw opening, a head-extension movement nearly synchronized with the opening movement in the lower jaw acceleration, and dorsal-neck muscle activities accompanying the synchronized movement were observed. The peak timing of these neck-muscle activities preceded the latencies of the stretch-reflex activities observed in the jaw-depressed tasks, but no anticipatory activities were observed in the dorsal-neck muscles. We conclude that neither the anticipatory activities nor the reflex activities observed in the passive depressions have effects on the initial part of the dorsal-neck muscle activities, which are related to the head-extension synchronized with the voluntary lower-jaw opening movement.

Propagation of the impact on the tooth caused by jaw tapping movement.

The influence of mechanical stimulation on the human body is extremely important. We hypothesized that if tooth impact is propagated to other sites of the body, this impact will have some effect on those sites as well. The purpose of this study was to investigate the extent to which tooth impact was propagated in the head and neck. It was found that the waves recorded on the upper canine are divided into a high frequency component and a low frequency component at a border of approximately 7 kHz. The amplitude of the impulse wave was 80.813 g for the low frequency component, and 177.839 g for the high frequency component. In terms of propagated vibration from the canine, the amplitude of the low frequency component was larger than that of the high frequency component, and greatest at the chin, followed in descending order by the zygomatic bone, forehead and vertebra prominens. For both frequency components, the amplitude of the propagated vibrations was small compared with the impulse waves. These results provide a basis for future analysis of the influence of such impact on cell response.

Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes.

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.

Effects of calmodulin antagonist (W-7) on phorbol ester (PMA)-induced contractile response in isolated rat aorta.

The aim of this study was to investigate effects of calmodulin antagonist (W-7) on the contractile response of the rat aorta induced by activation of protein kinase C (PKC) by phorbol ester. Phorbol 12-myristate 13-acetate (PMA) produced biphasic contraction i.e., a sustained contraction (initial contraction) and 17.9 +/- 1.7 min later, this progressively developed contraction was changed to a delayed contraction superimposed on the initial contraction. The delayed contraction was completely inhibited by treatment with nicardipine. The onset of the delayed contraction was significantly delayed by treatment with W-7, whereas same concentration of W-7 showed a weak relaxant effect (10%) on the PMA-induced maximal contraction of aorta. Higher concentration of W-7 strongly inhibited PMA-induced sustained contraction. These results suggest that PMA-induced biphasic contractile response may be regulated by calmodulin.

Chemotaxonomy on the leaf constituents of Thujopsis dolabrata Sieb. et Zucc.-Analysis of neutral extracts (diterpene hydrocarbon).

The leaf terpenes in Thujopsis dolabrata from all regions in Japan were analyzed using GC and GC-MS. The results show that the major constituents of the diterpene hydrocarbon fraction in this conifer are dolabradiene, hibaene, rimuene, 13-epi-dolabradiene and abietatriene. There were wide variations in the contents of the constituents among individuals and habitats. The results also show that T. dolabrata trees from 34 habitats can be classified into three groups based on the composition of the leaf diterpene hydrocarbon.

Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene.

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (BphA2) subunits, a ferredoxin (FD(BphA3)), and a ferredoxin reductase (FDR(BphA4)). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 (alpha subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP, FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISP(TodC1BphA2), FD(BphA3), and FDR(BphA4) exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP(TodC1BphA2) and ISP(BphA1A2) were present as heterohexamers, whereas ISP(TodC1C2) was present as a heterotetramer. The molecular activity (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

Influence on force curve exerted by jaw tapping force.

The purpose of this study, which made use of visual biofeedback, was to determine how methods of regulating jaw tapping force differed depending on the strength of the tapping, using the force curve as an index. Nine healthy examinees were asked to make 30-35 jaw tapping movements, reproducing the defined target tapping force as accurately as possible. We measured the duration of the tooth contact phase, the time to peak force, the first time derivative of force (peak dF/dt), and the time to peak dF/dt. The results indicated that the duration of the tooth contact phase and the time to peak force increased with the target value (P < 0.01). As the target rose, the peak dF/dt increased significantly (P < 0.01), but the time to peak dF/dt was not significant (P=0.134). We found that the higher the target value, the greater the degree of dependency on feedback information. We also found that both the peak dF/dt and the time to peak dF/dt were determined for each examinee prior to movement.

Emergence of multifunctional oxygenase activities by random priming recombination.

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.

[Copper supplement with cocoa for copper deficiency in patients with long-term enteral nutrition].

Copper deficiency (normal serum copper level: 78-136 micrograms/dl) has been reported in patients with long-term enteral nutrition, caused by a copper deficit in enteral nutrition. Occasionally, this leads to anemia and leukopenia. We used Hershey's pure cocoa that is rich in copper (content 3.8 mg/cocoa 100 g) for copper deficiency. A total of 86 (40 men and 46 women, mean age 69 years) patients on enteral nutrition were studied. The primary diseases were cerebral vascular disease in 71 patients, neurological disease in 5 and others in 10. Those who showed serum copper levels of 20 micrograms/dl or less (N = 8) were given 30-45 g of cocoa (copper content 1.14-1.71 mg) per day for about 40 days. Among them, two patients could not continue because of vomiting and diarrhea and were excluded from this study. Mean serum copper levels increased from 8.7 +/- 6.2 to 99.0 +/- 25.4 micrograms/dl (N = 6). Those who showed serum copper levels 20-77 mg/dl (N = 31) were given 10 g of cocoa (copper content 0.38 mg) per day for about 40 days. When mean serum copper levels increased from 50.5 +/- 19.3 to 89.0 +/- 12.9 micrograms/dl with cocoa administration, anemia and neutropenia caused by copper deficiency showed a tendency to improve. After completing the study period, cocoa was reduced to 5 g (copper content 0.19 mg) per day in 23 patients. The mean serum copper levels increased from 90.7 +/- 10.4 to 100.6 +/- 17.1 micrograms/dl for about 100 days. Recently, the amount of daily copper requirement for adults has been reported to be 1.28-2.5 mg per day. We showed that 10 g of cocoa (0.6 mg total copper: 0.38 mg in cocoa and 0.22 mg in other nutrients) is sufficient to treat copper deficiency, and 5 g of cocoa (0.37 mg total copper: 0.19 mg in cocoa and 0.18 mg in other nutrients) is enough to maintain the normal level of serum copper in patients with long-term enteral nutrition.

The enhancing effect of soybean-derived sterylglucoside and beta-sitosterol beta-D-glucoside on nasal absorption in rabbits.

The aim of this study was to elucidate the efficiency of soybean-derived sterylglucoside (SG) and its main component beta-sitosterol beta-D-glucoside (Sit-G), as nasal absorption enhancers. Nasal administration of verapamil with SG and Sit-G showed the higher bioavailabilities (60.4 and 90.7%, respectively) than that with lactose (39.8%). It was clear that SG and Sit-G promoted the absorption of verapamil through nasal mucosa. To elucidate the mechanism, we measured the calcein leakage from liposomes by incubation with SG, Sit-G, oleic acid, soybean-derived sterol, and beta-sitosterol to investigate transcellular absorption and measured the changes in intracellular Ca2+ concentrations ([Ca2+]i) by Sit-G to analyze paracellular absorption. The large amount of calcein leakage induced by enhancers was consistent with an enhancement of bioavailability of verapamil and insulin following nasal administration (oleic acid < SG < Sit-G). Moreover, Sit-G increased [Ca2+]i in the medium containing Ca2+, but not in Ca2+ free medium. This result suggested that Sit-G increases the fluidity of the mucosal membrane and facilitates Ca2+ influx from extracellular sources. In conclusion, a possible explanation for SG and Sit-G to promote drug absorption, is that they may affect both paracellular pathway and transcellular pathways caused by pertubation of lipid.

Regulation of human jaw tapping force with visual biofeedback.

The purpose of this study, which made use of visual biofeedback, was to determine whether jaw tapping force reproduction is related to the strength of tapping and to investigate how jaw tapping force affects the tapping movement curve. Nine healthy examinees were asked to reproduce jaw tapping force. We found that the ease and method of regulating jaw tapping force differed depending on the target force. We also found that jaw tapping force was regulated by alteration of the jaw opening distance, the duration of the tooth contact phase, the duration of the jaw closing phase, the maximum jaw opening velocity, and the maximum jaw closing velocity. However, the duration of the jaw opening phase and cycle time was not affected by force regulation under our experimental conditions.

Management of suspected nosocomial infection: an audit of 19 hospitalized patients with septicemia caused by Bacillus species.

From April to August of 2000, Bacillus spp. were detected in the blood culture of 29 patients in a hospital in Japan. Of these patients, 19 had clinical signs of septicemia; positive culture in the remaining 10 patients was attributed to contamination with skin flora at the site of puncture. Of the 18 strains evaluated, 15 were Bacillus cereus, 2 were Bacillus subtilis, and one was Bacillus licheniformis. The only hospital death observed was that of a patient who had no clinical signs of septicemia at the time of blood sampling. That death is now considered attributable to the underlying neoplasm. The hospital committee for prevention of nosocomial infection concluded after a critical review of the patient records that the cause of septicemia in most cases had been contaminated intravenous lines. To control the situation, the committee recommended the use of a new skin disinfectant, and medical personnel were advised to avoid infusion pauses with interruption of intravenous lines and to replace the caps for the stopcocks with new ones each time the caps were removed. These measures were rigorously observed in addition to the conventional measures for preventing catheter sepsis, and the incidence of septicemia due to the Bacillus spp. declined dramatically thereafter.

Alpha-adrenoceptor agonists produce Ca2+ oscillations in isolated rat aorta: role of protein kinase C.

We investigated the relationship between tension development and the cytosolic free Ca2+ level ([Ca2+]i) in responses to norepinephrine (NE) and selective alpha2-adrenoceptor agonist, UK14,304 of the endothelium-denuded rat aorta loaded with fura PE-3. NE (3 x 10(-8) M) evoked a rapid increase in [Ca2+]i followed by slight decreasing to a steady state level and produced a contraction. After the NE-induced increase in [Ca2+]i had reached a maximum, the [Ca2+]i showed persistent oscillations. The Ca2+ oscillations were superimposed on the sustained increase in [Ca2+]i. UK14,304 (3 x 10(-6) M) also evoked an increase in [Ca2+]i and produced a contraction. However, the UK14,304-induced effect on [Ca2+]i was characterized by pronounced oscillations, and the amplitude of the sustained increase in [Ca2+]i was less than that seen with NE. Protein kinase C inhibitor, Ro31-8220 (3 x 10(-6) M) and verapamil (10(-5) M) abolished both NE and UK14,304-evoked Ca2+ oscillations. UK14,304-induced contractions were also strongly inhibited by Ro31-8220 and verapamil. However, NE induced contractions were partly inhibited by these inhibitors. The sustained increases in [Ca2+]i evoked NE and UK14,304 were not significantly inhibited by Ro31-8220 and verapamil. These results suggest that NE and UK14,304 produce Ca2+ oscillations during sustained contractions in rat aorta. The alpha2 adrenoceptor agonist, UK14,304-induced sustained contraction and Ca2+ oscillations may be due to PKC activation and opening of voltage-dependent L type Ca2+ channels.