Identification of the Novel Tooth-Specific Transcription Factor AmeloD.

Basic-helix-loop-helix (bHLH) transcription factors play an important role in various organs' development; however, a tooth-specific bHLH factor has not been reported. In this study, we identified a novel tooth-specific bHLH transcription factor, which we named AmeloD, by screening a tooth germ complementary DNA (cDNA) library using a yeast 2-hybrid system. AmeloD was mapped onto the mouse chromosome 1q32. Phylogenetic analysis showed that AmeloD belongs to the achaete-scute complex-like ( ASCL) gene family and is a homologue of ASCL5. AmeloD was uniquely expressed in the inner enamel epithelium (IEE), but its expression was suppressed after IEE cell differentiation into ameloblasts. Furthermore, AmeloD expression showed an inverse expression pattern with the epithelial cell-specific cell-cell adhesion molecule E-cadherin in the dental epithelium. Overexpression of AmeloD in dental epithelial cell line CLDE cells resulted in E-cadherin suppression. We found that AmeloD bound to E-box cis-regulatory elements in the proximal promoter region of the E-cadherin gene. These results reveal that AmeloD functions as a suppressor of E-cadherin transcription in IEE cells. Our study demonstrated that AmeloD is a novel tooth-specific bHLH transcription factor that may regulate tooth development through the suppression of E-cadherin in IEE cells.

Development of an improved RT-PCR for specific detection of spring viraemia of carp virus.

Spring viraemia of carp (SVC) is a rhabdovirus infection, which has a significant economic impact in pond cultures of carp in Europe and western Independent States of the former Soviet Union. The causative agent of SVC, spring viraemia of carp virus (SVCV), has been divided into four subgroups, Ia, Ib, Ic and Id, on the basis of glycoprotein (G) protein gene sequences. In this study, a new primer set was designed from a G gene sequence of SVCV to identify the four subtypes of SVCV by reverse transcription polymerase chain reaction (RT-PCR). The specific PCR products of 369 bp were amplified from 15 SVCV isolates of all four subtypes. However, pike fry rhabdovirus (PFRV), which is antigenically related to SVCV, and other viruses antigenically related to SVCV and PFRV were not amplified. The four subtypes of SVCV were specifically amplified by the RT-PCR. Furthermore, the detection limit of the RT-PCR was 7.1 × 10(2) copies/reaction, and it was not influenced by the addition of RNA extracted from fish tissues. The RT-PCR will be applied not only to RNA extracted from viral suspensions, but also from fish tissue. It will contribute to rapid identification of SVCV in fish with clinical signs of SVC.

Pathogenesis of acute and chronic diseases caused by cyprinid herpesvirus-3.

The pathogenesis of cyprinid herpesvirus-3 (CyHV-3) was studied using different lineages of carp/koi. After exposure to the virus, infected cells were first found in the skin by histopathology and by in situ hybridization. The epidermis of the skin was most severely damaged and often sloughed off in the fish sampled on days 5 through 8, and the fish that were highly sensitive to the virus died within 8 or 10 days after infection. Serum osmolality of the infected fish, particularly just before death, was significantly lower, suggesting that the osmotic shock consequent on the damage to the skin was the direct cause of the acute deaths. On the other hand, clinical and histopathological observations indicate that the carp of a less sensitive lineage most probably died of viral encephalitis around 3 weeks after infection. For these fish, the largest number of infected cells was found in the central nervous system (CNS) sampled on day 12. A substantial amount of viral genome was found in the CNS of carp surviving more than 1 year after the infection. Thus, the CNS is probably a major target for CyHV-3, and the virus can persistently infect the CNS, presumably establishing latency.

A new method for displaying the lingual artery using high-resolution three-dimensional phase-contrast magnetic resonance angiography.

The aim of this study was to display the lingual artery superimposed on the anatomical image and to confirm its course and relation to the adjacent structures, noninvasively. Nineteen volunteers participated in the magnetic resonance imaging (MRI) study and one was excluded for excessive movement during scanning. A three-dimensional phase-contrast sequence (3D-PC) of magnetic resonance angiography (MRA) was used for vessel images, and a 3D-T1 high-resolution volume examination (THRIVE) was used for anatomical images. Colour-coded vessel images from 3D-PC MRA were superimposed on the 3D volume anatomical images, and the arterial course and relation to the adjacent structures were confirmed with multiplanar reconstructed cross-sectional (MPR) images. 3D-PC MRA images visualized the lingual artery in all 18 subjects and the sublingual artery in 14 subjects. In seven of 18 cases the bilateral sublingual arteries were shown to run side by side but had no contact with the sublingual veins. They ran together with the sublingual veins in four cases. Three cases showed irregular patterns. The bilateral sublingual arteries could not be identified in four cases. 3D-PC MRA images of the lingual artery superimposed on the anatomical images may be clinically useful to confirm its course and relationship to the adjacent structures before surgery, in order to prevent haemorrhage.

Quantitative evaluation of vascularity within cervical lymph nodes using Doppler ultrasound in patients with oral cancer: relation to lymph node size.

OBJECTIVES: The aim of this study was to quantitatively evaluate the relationship between vascularity within lymph nodes and lymph node size on Doppler ultrasound images of patients with oral cancer. METHODS: A total of 310 lymph nodes (86 metastatic, 224 benign) from 63 patients with oral cancer were classified into 4 groups according to their short axis diameters: Group 1, short axis diameters of 4-5 mm; Group 2, 6-7 mm; Group 3, 8-9 mm; and Group 4, ≥ 10 mm. Vascular and scattering indices of lymph nodes on Doppler ultrasound images were analysed quantitatively. The vascular index was defined as the ratio of blood flow area to the whole lymph node area and the scattering index was defined as the number of isolated blood flow signal units. RESULTS: For metastatic lymph nodes, the vascular index was highest in Group 1 and decreased as lymph node size increased. The vascular index of benign lymph nodes did not differ significantly among the four groups. The vascular index of metastatic lymph nodes was significantly higher than that of benign lymph nodes in Group 1. For metastatic lymph nodes, the scattering index increased as lymph node size increased and was significantly higher than that of benign lymph nodes in Groups 2-4. CONCLUSIONS: An increase in vascularity is a characteristic of Doppler ultrasound findings in small metastatic lymph nodes. As the metastatic lymph node size increases, blood flow signals become scattered, and the scattering index increases.

Evaluation of T2 values and apparent diffusion coefficient of the masseter muscle by clenching.

OBJECTIVE: The aim of this study was to evaluate the changes in T2 values and apparent diffusion coefficient (ADC) in the masseter muscle by clenching in healthy volunteers. METHODS: 37 volunteers were enrolled in the study. We measured bite force using pressure-sensitive paper and a T2 map. The ADC map was obtained at rest, during clenching, immediately after and 5 min after clenching. The spin-echo sequence was used to calculate T2, and single-shot spin-echo echo planar imaging was used to calculate the ADC. The motion-probing gradients (MPGs) were applied separately along the posterior-to-anterior (PA), right-to-left (RL) and superior-to-inferior (SI) directions, with b values of 0, 300 and 600 s mm(-2) in each direction. ADC-PA, ADC-RL, and ADC-SI values were obtained, and we calculated the ADC-iso for the mean diffusivity. RESULTS: There were no significant differences between the stronger and weaker sides of bite force before, during or 5 min after clenching for T2 and ADC. The bite force had little effect on these parameters; thus, we used the average of the two sides for the following analyses. Time course analysis of ADC-iso, ADC-PA, ADC-RL and ADC-SI demonstrated a marked increase after clenching and a rapid decrease immediately after clenching, although they did not completely return to the initial values; however, the change in ADC-RL was significantly greater than those in ADC-PA or ADC-SI (P<0.001 each). The changes in T2 were similar to those of ADC, although not as marked. CONCLUSIONS: ADC (especially ADC-RL) was altered by contraction of the masseter muscle.

Distribution and species composition of juvenile and adult scombropids (Teleostei, Scombropidae) in Japanese coastal waters.

Two scombropid fishes, Scombrops boops and Scombrops gilberti, are closely related and commercially important species in Japan. These species are often confused in commercial markets because of their morphological similarity. In this study, scombropid specimens collected from various Japanese coastal waters were subjected to polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and phylogenetic analysis of the 16S rRNA gene in mitochondrial DNA. These analyses showed that all the scombropid specimens collected from localities in the Sea of Japan were identified as S. boops, whereas those from the Pacific Ocean included two species, S. boops and S. gilberti. Almost all juvenile (<200 mm standard body length, S(L)) S. gilberti originated from the Pacific coastal waters of the northern Japan, whereas adults (>400 mm S(L)) were found only in deep water off the Izu Peninsula to the Izu Islands. This suggests that S. gilberti might migrate extensively during its life cycle. In addition, differences in the number of specimens and the distribution between the two species suggest that S. gilberti is less abundant than S. boops in Japanese waters.

Genotyping of Edwardsiella ictaluri isolates in Japan using amplified-fragment length polymorphism analysis.

AIM: The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007. METHODS AND RESULTS: Ten isolates of Edw. ictaluri in 2007-2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles. CONCLUSION: No DNA polymorphisms were found among Japanese Edw. ictaluri isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish.

AIMS: We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture. METHODS AND RESULTS: In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l-arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate. CONCLUSIONS: Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described. SIGNIFICANCE AND IMPACT OF THE STUDY: The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.

Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products.

Using murine models, we have previously demonstrated that recombinant adeno-associated virus (rAAV)-mediated microdystrophin gene transfer is a promising approach to treatment of Duchenne muscular dystrophy (DMD). To examine further therapeutic effects and the safety issue of rAAV-mediated microdystrophin gene transfer using larger animal models, such as dystrophic dog models, we first investigated transduction efficiency of rAAV in wild-type canine muscle cells, and found that rAAV2 encoding beta-galactosidase effectively transduces canine primary myotubes in vitro. Subsequent rAAV2 transfer into skeletal muscles of normal dogs, however, resulted in low and transient expression of beta-galactosidase together with intense cellular infiltrations in vivo, where cellular and humoral immune responses were remarkably activated. In contrast, rAAV2 expressing no transgene elicited no cellular infiltrations. Co-administration of immunosuppressants, cyclosporine and mycophenolate mofetil could partially improve rAAV2 transduction. Collectively, these results suggest that immune responses against the transgene product caused cellular infiltration and eliminated transduced myofibers in dogs. Furthermore, in vitro interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more susceptibly than murine ones. Our results emphasize the importance to scrutinize the immune responses to AAV vectors in larger animal models before applying rAAV-mediated gene therapy to DMD patients.

Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscular disorder caused by a defect in the DMD gene. AAV vector-mediated micro-dystrophin cDNA transfer is an attractive approach to treatment of DMD. To establish effective gene transfer into skeletal muscle, we examined the transduction efficiency of an AAV vector in skeletal muscles of dystrophin-deficient mdx mice. When an AAV vector encoding the LacZ gene driven by a CMV promoter (AAV-CMVLacZ) was introduced, beta-galactosidase expression markedly decreased in mdx muscle 4 weeks after injection due to immune responses against the transgene product. We also injected AAV-CMVLacZ into skeletal muscles of mini-dystrophin-transgenic mdx mice (CVBA3'), which show ameliorated phenotypes without overt signs of muscle degeneration. AAV vector administration, however, evoked substantial immune responses in CVBA3' muscle. Importantly, AAV vector using muscle-specific MCK promoter also elicited responses in mdx muscle, but at a considerably later period. These results suggested that neo-antigens introduced by AAV vectors could evoke immune reactions in mdx muscle, since increased permeability allowed a leakage of neo-antigens from the dystrophin-deficient sarcolemma of muscle fibers. However, resident antigen-presenting cells, such as myoblasts, myotubes and regenerating immature myofibers, might also play a role in the immune response.

Volume changes in human masticatory muscles between jaw closing and opening.

Most jaw muscles are complex, multipennate with multiple components. The morphologic heterogeneity of masticatory muscles reflects their functions. We hypothesized that the volume of masticatory muscles changes between jaw closing and opening, and that there is a difference in the volume change among the muscles. Magnetic resonance images of the entire head were obtained in ten normal young adult subjects before and after maximum jaw opening. The volume changes of the masseter, medial, and lateral pterygoid muscles were measured. Only slight changes were seen in the masseter and medial pterygoid muscles. The lateral pterygoid muscle, however, significantly decreased its volume during jaw opening. The results provide normative values of muscle volume in living subjects, and suggest that the volume changes differ among jaw muscles.

Organ specificity of a vacuolar Ca2+-binding protein RVCaB in radish and its expression under Ca2+-deficient conditions.

Radish vacuoles contain a new type of Ca2+-binding protein (RVCaB) with high capacity and low affinity for Ca2+. The protein is able to stimulate Ca2+ uptake into vacuoles, which is driven by Ca2+-ATPase and Ca2+/H+ antiporter. In the present study, we found that the level of RVCaB mRNA is high in seedling hypocotyls and mature taproots but low in young roots and mature leaves. The RVCaB protein was abundant in hypocotyls and taproots but absent in leaves. The levels of the transcript and protein of RVCaB in taproots were gradually increased during maturation. The level of RVCaB mRNA in seedling hypocotyls doubled within a few hours when the growth medium was changed from 10 mM CaCl2 to water, although the level was strongly suppressed in 100 mM CaCl2. This response of the RVCaB gene was specific to Ca2+ and did not occur with other ions including K+ and Mg2+. RVCaB functioning as a Ca2+-sequestering protein in taproot vacuoles to provide for the Ca2+ deficiency is discussed.

Pro-apoptotic effect of presenilin 2 (PS2) overexpression is associated with down-regulation of Bcl-2 in cultured neurons.

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimer's disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus-mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild-type or mutant (N141I) PS2-expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control beta-galactosidase-expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine-induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild-type counterpart. Staurosporine-induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl-2 protein expression was specifically down-regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C- and N-terminal fragments of PS2. Additionally, expression of mutant, but not wild-type PS2, increased the production of beta-amyloid protein (Abeta) 42. These data collectively suggest that the pro-apoptotic effect of PS2 is mediated by down-regulation of Bcl-2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.

[Intramedullary spinal cord metastasis of lung adenocarcinoma causing Brown-Séquard Syndrome].

A 36-year-old woman admitted to our hospital because of numbness in the left limbs and weakness in the right arm, and was subsequently given a diagnosis of intramedullary spinal cord metastases from lung cancer. The patient had lung adenocarcinoma with metastases to the brain, spine and lymph nodes. Occipital craniotomy, radiation therapy and chemotherapy were performed on the lesions in the year following June 1994. In June 1995, however, she complained of numbness in the left limbs and weakness in the right arm. Compatible with her neurological manifestation, MRI demonstrated tumors in the right side of the cord at the spinal level of C3-4 and C7-Th1, both of which were of high density in T2-enhanced conditions with enhancement by gadolinium-diethylenetriamine pentaacetic acid. No invasion from spinal metastasis was detected by CT, scintigraphy or MRI. We therefore diagnosed her manifestation as Brown-Séquard syndrome caused by intramedullary spinal cord metastatic tumors of lung adenocarcinoma. In order to avoid paraplegia and dysfunction of the bladder and bowel, radiation therapy of the cord lesions with total dose of 44 Gy was performed. Her neurologic manifestation was improved, restoring her quality of life, as the tumor size estimated by MRI decreased. Four months later, however, she died of lung adenocarcinoma that developed accompanied with severe peritonitis carcinomatosa and multiple metastases.

Decreased sensory cortical excitability after 1 Hz rTMS over the ipsilateral primary motor cortex.

OBJECTIVES: To study changes in the excitability of the sensory cortex by repetitive transcranial magnetic stimulation (rTMS) in humans. METHODS: Somatosensory evoked potentials (SEPs) and antidromic sensory nerve action potentials (SNAPs) were elicited by right median nerve stimulation at the wrist before and after low frequency (1 Hz) rTMS over the left motor cortex, lateral premotor cortex, sensory cortex, and also after sham stimulation. The intensity of rTMS was fixed at 1.1 times the active motor threshold at the hand area of motor cortex. RESULTS: N20 peak (N20p)-P25 and P25-N33 amplitudes were suppressed after rTMS over the motor cortex, whereas the N20 onset (N20o)-N20p and SNAP amplitudes were not affected. They recovered to the baseline about 100 min after the rTMS. rTMS over the premotor cortex or sensory cortex or sham stimulation had no suppressive effect on SEPs. CONCLUSIONS: The reduction of N20p-P25 and P25-N33 components without any changes of N20o-N20p amplitude suggests that the suppression occurs in the sensory cortex. rTMS (1 Hz) of the motor cortex induces a long-lasting suppression of the ipsilateral sensory cortex even at an intensity as low as 1.1 times the active motor threshold, probably via cortico-cortical pathways between motor and sensory cortex.

Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A): comparison of rat and human PDE11A splicing variants.

We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3-1.6 and 2.1-3.9 microM, respectively), the Vmax values differed significantly (RNPDE11A4 >> RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 >> HSPDE11A2 > HSPDE11A3 >> HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants.

Effects of selenium deficiency on expression of selenoproteins in bovine arterial endothelial cells.

Damage to the vascular endothelium by reactive oxygen species causes many cardiovascular diseases including atherosclerosis. Such damage can be prevented by selenium (Se), which is thought to exert its actions mainly through the expression of selenoproteins. Se deficiency increased the susceptibility to tert-butylhydroperoxide (t-BuOOH) and enhanced lipid peroxidation in bovine arterial endothelial cells (BAEC). We investigated the effects of Se deficiency on the expression of the selenoproteins in BAEC. 75Se metabolic labeling analysis and RT-PCR analysis revealed that BAEC expressed two glutathione peroxidase (GPx) isozymes, cytosolic GPx (cGPx) and phospholipid hydroperoxide GPx (PHGPx), three thioredoxin reductase (TrxR) isozymes, TrxR1, TrxR2 and TrxR3, and selenoprotein P (SelP). Se deficiency reduced both enzyme activity and mRNA level of cGPx, but did not affect those of PHGPx. SelP mRNA level was also reduced by Se deficiency, although the extent of reduction was much smaller than that of cGPx mRNA. We further found that TrxR activity was also decreased by Se deficiency but none of the mRNA levels of TrxR isozymes were reduced. These results indicate that vascular endothelial cells express several selenoproteins including cGPx, PHGPx, TrxR isozymes and SelP which might play important roles in the defense system against oxidative stresses and that the expressions of these selenoproteins are differently regulated by Se status.

Integration of a picture archiving and communication system with videocapture and computed radiography in a dental hospital.

A method for the integration of a picture archiving and communication system (PACS) at Kyushu University Dental Hospital with radiological information (RIS) and hospital information (HIS) systems is described. CT, US and DSA from different manufacturers were integrated by videocapture and then subsequently integrated with computed radiography (CR) by means of DICOM. The approximate amount of data stored each month on optical discs is 2 GB. The system does not incorporate intra-oral radiography.

Interhemispheric interaction between the hand motor areas in patients with cortical myoclonus.

OBJECTIVE: To study interhemispheric interaction between the hand motor areas of both hemispheres through the corpus callosum in myoclonus epilepsy. SUBJECTS: Five patients with benign myoclonus epilepsy and ten age matched normal volunteers. METHODS: We studied effects of a medially directed conditioning stimulus over the right hand motor area on responses in the right first dorsal interosseous muscle to a posteriorly directed test stimulus over the left hand motor area. RESULTS: In normal subjects, inhibition was evoked at interstimulus intervals (ISIs) of 8-20ms (late inhibition). In contrast, facilitation occurred in patients at ISIs of 4-6ms (early facilitation) with no late inhibition. CONCLUSIONS: The lack of late inhibition in the patients is consistent with the idea that cortical inhibitory interneurones are affected in myoclonus epilepsy. We propose that this releases interhemispheric facilitation from powerful surround inhibition. The consequence is a predominant early facilitation between the hemispheres in patients with myoclonus epilepsy.