Abstract: Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.

Abstract: We visualized respiratory neuron activity covering the entire ventral medulla using optical recordings in a newborn rat brainstem-spinal cord preparation stained with voltage-sensitive dye. We measured optical signals from several seconds before to several seconds after the inspiratory phase using the inspiratory motor nerve discharge as the trigger signal; we averaged the optical signals of 50-150 respiratory cycles to obtain an optical image correlating particularly to inspiratory activity. The optical images we obtained from the ventral approach indicated that neuron activity first appeared during the respiratory cycle in the limited region of the rostral ventrolateral medulla (RVLM), preceding the onset of inspiratory activity by approximately 500 msec. During the inspiratory phase, plateau activity appeared in the more caudal ventrolateral medulla at the level of the most rostral roots of the XIIth nerve. Comparison with electrophysiological recordings from respiratory neurons in the RVLM suggested that the optical signals preceding the inspiratory burst reflect preinspiratory neuron activity in this area. This RVLM area was determined to be ventrolateral to the facial nucleus and close to the ventral surface. We referred to this functional neuron group as the para-facial respiratory group (pFRG). Partial, bilateral electrical lesioning of the pFRG significantly reduced the respiratory frequency, together with changes in the spatiotemporal pattern of respiratory neuron activity. Our findings suggest that the pFRG comprises a neuronal population that is involved in the primary respiratory rhythm generation in the rostrocaudally extending respiratory neuron network of the medulla.

Abstract: The hypothalamus regulates energy intake by integrating the degree of starvation or satiation with the status of the environment through a variety of neuronal and blood-derived signals. Ghrelin, a peptide produced in the stomach and hypothalamus, stimulates feeding and GH secretion. Centrally administered ghrelin exerts an orexigenic activity through the neuropeptide Y (NPY) and agouti-related protein systems. The interaction between ghrelin and other hypothalamic orexigenic peptides, however, has not been clarified. Here, we investigated the anatomical interactions and functional relationship between ghrelin and two orexigenic peptides, orexin and melanin-concentrating hormone (MCH), present in the lateral hypothalamus. Ghrelin-immunoreactive axonal terminals made direct synaptic contacts with orexin-producing neurons. Intracerebroventricular administration of ghrelin induced Fos expression, a marker of neuronal activation, in orexin-producing neurons but not in MCH-producing neurons. Ghrelin remained competent to induce Fos expression in orexin-producing neurons following pretreatment with anti-NPY IgG. Pretreatment with anti-orexin-A IgG and anti-orexin-B IgG, but not anti-MCH IgG, attenuated ghrelin-induced feeding. Administration of NPY receptor antagonist further attenuated ghrelin-induced feeding in rats treated with anti-orexin-IgGs. Ghrelin-induced feeding was also suppressed in orexin knockout mice. This study identifies a novel hypothalamic pathway that links ghrelin and orexin in the regulation of feeding behavior and energy homeostasis.

Abstract: Only 15% to 20% of patients with chronic hepatitis C have a sustained virological response to interferon monotherapy. The aim of the present study was to compare the efficacy and safety of interferon, in combination with oral cyclosporin A, with interferon monotherapy in the treatment of chronic hepatitis C. Methods. We assigned 120 patients with chronic hepatitis C to receive the standard Japanese dose of interferon α2b alone for 24 weeks or that dose of interferon α2b in combination with cyclosporin A, at doses of 200 mg daily for the first 4 weeks and 100 mg daily for the following 20 weeks. All patients were assessed for drug safety, tolerance, and efficacy at the end of weeks 4, 12, 24, and 48. Efficacy was assessed by the disappearance of serum hepatitis C virus (HCV)-RNA by polymerase chain reaction and normalization of serum aminotransferase. The primary endpoint was a sustained virological response; i.e., sustained undetectable serum HCV RNA at 48 weeks. Results. The sustained virological response rate was significantly higher in the combination therapy group (42/76) than in the monotherapy group (14/44; P = 0.01). The sustained biochemical response rate was also higher in the combination therapy group (46/76) than in the monotherapy group (17/44; P = 0.017). In patients with genotype 1 and high viral loads, the sustained virological response rate was markedly higher in the combination therapy group (16/38) than in the monotherapy group (1/21; P = 0.006). Side-effect profiles were similar in the two groups. Conclusions. In patients with chronic hepatitis C; combined interferon and cyclosporin A treatment was more effective than interferon monotherapy. The benefit was mostly achieved in patients with a high viral load and HCV genotype 1.

Abstract: Peptide YY3-36 (PYY) has emerged as an important signal in the gut-brain axis, with peripherally administered PYY affecting feeding and brain function. For these effects to be direct, PYY would have to cross the blood-brain barrier (BBB). Here, we determined the permeability of the BBB to PYY radioactively labeled with 131I (I-PYY). Multiple-time regression analysis showed the unidirectional influx rate (Ki) from blood-to-brain for I-PYY to be 0.49 +/- 0.19 microl/g-min, a rate similar to that previously measured for leptin. Influx was not inhibited by 1 microg/mouse of unlabeled PYY, suggesting PYY crosses the BBB by transmembrane diffusion. About 0.176% of the i.v.-injected dose of I-PYY was taken up by brain, an amount similar to that for other peptides important in gut-brain communication. Capillary depletion showed that 69% of I-PYY crossed the BBB to enter the parenchymal space of the brain, and high-performance liquid chromatography demonstrated that the radioactivity in this space represented intact I-PYY. After intracerebroventricular injection, I-PYY crossed from brain to blood by the mechanism of bulk flow. We conclude that PYY crosses in both the blood-to-brain and brain-to-blood directions by nonsaturable mechanisms. Passage across the BBB provides a mechanism by which blood-borne PYY can affect appetite and brain function.

Abstract: The differentiation and functions of osteoclasts (OC) are regulated by osteoblast-derived factors such as receptor activator of NFKB ligand (RANKL) that stimulates OC formation, and a novel secreted member of the TNF receptor superfamily, osteoprotegerin (OPG), that negatively regulates osteoclastogenesis. In examination of the preosteoclast (pOC) culture, pOCs formed without any additives expressed tartrate-resistant acid phosphatase (TRAP), but showed little resorptive activity. pOC treated with RANKL became TRAP-positive OC, which expressed intense vacuolar-type H(+)-ATPase and exhibited prominent resorptive activity. Such effects of RANKL on pOC were completely inhibited by addition of OPG. OPG inhibited ruffled border formation in mature OC and reduced their resorptive activity, and also induced apoptosis of some OC. Although OPG administration significantly reduced trabecular bone loss in the femurs of ovariectomized (OVX) mice, the number of TRAP-positive OC in OPG-administered OVX mice was not significantly decreased. Rather, OPG administration caused the disappearance of ruffled borders and decreased H(+)-ATPase expression in most OC. OPG deficiency causes severe osteoporosis. We also examined RANKL localization and OC induction in periodontal ligament (PDL) during experimental movement of incisors in OPG-deficient mice. Compared to wild-type OPG (+/+) littermates, after force application, TRAP-positive OC were markedly increased in the PDL and alveolar bone was severely destroyed in OPG-deficient mice. In both wild-type and OPG-deficient mice, RANKL expression in osteoblasts and fibroblasts became stronger by force application. These in vitro and in vivo studies suggest that RANKL and OPG are important regulators of not only the terminal differentiation of OC but also their resorptive function. To determine resorptive functions of OC, we further examined the effects of specific inhibitors of H(+)-ATPase, bafilomycin A1, and lysosomal cysteine proteinases (cathepsins), E-64, on the ultrastructure, expression of these enzymes and resorptive functions of cultured OC. In bafilomycin A1-treated cultures, OC lacked ruffled borders, and H(+)-ATPase expression and resorptive activity were significantly diminished. E-64 treatment did not affect the ultrastructure and the expression of enzyme molecules in OC, but significantly reduced resorption lacuna formation, by inhibition of cathepsin activity. Lastly, we examined the expression of H(+)-ATPase, cathepsin K, and matrix metalloproteinase-9 in odontoclasts (OdC) during physiological root resorption in human deciduous teeth, and found that there were no differences in the expression of these molecules between OC and OdC. RANKL was also detected in stromal cells located on resorbing dentine surfaces. This suggests that there is a common mechanism in cellular resorption of mineralized tissues such as bone and teeth.

Abstract: Chemokines play an essential role in the progression of rheumatoid arthritis (RA). In the present study we examined the expression and regulatory mechanisms of IFN-γ inducible protein (IP)-10 in RA synovitis. RA synovial fluid contained greater amounts of IP-10 than did synovial fluid from patients with osteoarthritis. Immunolocalization analysis indicated that IP-10 was associated mainly with infiltrating macrophage-like cells, and fibroblast-like cells in the RA synovium. The interaction of activated leukocytes with fibroblast-like synoviocytes resulted in marked increases in IP-10 expression and secretion. Moreover, induction of IP-10 was mediated via specific adhesion molecules, as indicated by the finding that both anti-integrin (CD11b and CD18) and intercellular adhesion molecule-1 antibodies significantly inhibited IP-10 induction. These results suggest that IP-10 expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leukocytes with fibroblast-like synoviocytes, and that IP-10 may contribute to the recruitment of specific subpopulations of T cells (Th1 type) from the bloodstream into the synovial joints.

Abstract: Although screening for stomach cancer is a widespread community service in Japan, the benefits of the screening program remain unclear. Our study investigated prospectively the relation between participation in stomach-cancer screening during the past 12 months and subsequent deaths. Data was derived from the Japan Collaborative Cohort Study, in which 480 stomach-cancer deaths were identified during an 8-year follow-up period. Cox proportional hazard regression was used to estimate the relative risk of death from stomach cancer and that from other causes while adjusting for potential confounding factors. In men, screening participation was associated significantly with a reduced risk of death from stomach cancer (relative risk [RR] = 0.54; 95% confidence interval [CI] = 0.41-0.70). The extent of the risk reduction was greater than potential health selection (for deaths other than stomach, RR = 0.71). The adjustment for potential confounding variables, however, attenuated the difference in risk of death (stomach cancer, RR = 0.65; other causes, RR = 0.71). In women, the magnitude of the association between screening participation and death from stomach cancer (RR = 0.74; 95% CI = 0.52-1.07) was equal to that for deaths from non-stomach cancers (RR = 0.74). Subgroup analysis showed that women with a parental history of stomach cancer had a reduced risk of death from stomach cancer associated with screening (RR = 0.32; 95% CI = 0.12-0.87). The present results underline the potential for selection bias in observational studies, and thus it remains an open question whether screening for stomach cancer is effective.

Abstract: Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 I¼g/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicate that EGCg may be a valuable therapeutic agent against H. pylori infection.

Abstract: Recent investigations have indicated the importance of secondary brain damage in the pathophysiology of intracerebral hemorrhage (ICH), which includes ischemic brain damage and edema formation around a hematoma. The purpose of the current study is to investigate chronological changes of perihematomal edema in patients with human ICH and also the correlation between volume of perihematomal edema and neurological status. Fourteen patients with medium-sized putaminal hemorrhage (29.4 ± 13.2 ml) without hematoma enlargement were included in this study. To investigate chronological changes of perihematomal edema, we performed CT scans prospectively on the day of hemorrhage and repeated them on days 1, 3, 7, 14, and 28. We evaluated the patients neurologically using the NIH stroke scale on the day a CT scan was performed. The volume of perihematomal edema in human ICH increased rapidly three days after hemorrhage and the score on the NIH stroke scale showed a deterioration. The volume of perihematomal edema then increased slowly until day 14 after hemorrhage, and decreased thereafter. Despite progression of perihematomal edema, the score on the NIH stroke scale improved gradually after day 3.

Abstract: Fractionated extracts of persimmon (Diospyros kaki) peels were studied for cytotoxic activity, multidrug resistance (MDR) reversal activity, anti-human immunodeficiency virus (HIV) activity and anti-Helicobacter pylori (H. pylori) activity. The potent cytotoxic activity against human oral squamous cell carcinoma cells (HSC-2) and human submandibular gland tumor (HSG) cells was found in the acetone fractions (A4 and A5) with IC(50) ranging from 21 to 59 micro g/mL. However, the cytotoxic activity was not correlated with the radical intensity of the fractions. Three 70% MeOH extract fractions (70M2-4) produced radical and efficiently scavenged the O(2)(-) produced by hypoxanthine and xanthine oxidase reaction. All of the fractions tested were not effective for anti-H. pylori and anti-HIV. Fractions H3 and H4 of hexane extract, and M2 and M3 of MeOH extract showed a remarkable MDR reversal activity comparable with that of (+/-)-verapamil (a positive control). These results indicate the therapeutic value of persimmon peel extracts as potential antitumor and MDR-reversing agents.

Abstract: SUMMARY In the present study, the compositional changes and knoop hardness of the cavity floor prepared by Er,Cr:YSGG laser irradiation was com­ pared with that of the conventional bur cavity. Fifteen laser and 15 bur cavities were cross-sec­ tioned, and subjected to atomic analysis by SEM­ EDX and knoop hardness test. Statistical analyses were performed using the Mann-Whitney U-test; a value of P < 0-01 was considered significant. Surface characteristics of the prepared cavities were also investigated by light microscopy and scanning electron microscopy (SEM). The results showed that the quantities of Ca (Ca weight %) and P (P weight %) were increased significantly in the laser cavity floor but no significant differences were found between the Ca/P ratio and knoop hardness number of laser and bur cavities. The SEM observation revealed that the lased cavity surface was irregular and there was also the absence of a smear layer; the orifice of dentinal tubules was exposed. Er,Cr:YSGG laser device is considered as one of the most effective and safe devices for cavity preparation because of its many advantages. This includes easy delivery system, minimal thermal damage to the surrounding tissues, minimal thermal-induced changes of dental hard tissue compositions, and

Abstract: Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.

Abstract: Background and Study Aims: Histological examination of gastrointestinal lesions is currently based on light-microscopic examination of thin-slice specimens, with hematoxylin and eosin staining. A study of the use of laser-scanning confocal microscopy (LCM) to obtain immediate microscopic images of untreated specimens for examining colorectal lesions was carried out. A probe-type LCM prototype endomicroscope that can be passed through the working channel of an endoscope has also been developed. Materials and Methods: The study materials consisted of colorectal lesions resected either endoscopically or surgically at Showa University Northern Yokohama Hospital. One hundred untreated specimens were examined using LCM. The histopathological findings in the lesions were seven cases of normal colonic mucosa, five hyperplastic polyps, 68 adenomas with low-grade dysplasia, 10 adenomas with high-grade dysplasia, and 10 adenocarcinomas. An argon laser beam with a wavelength of 488 nm was used for the LCM study. Observation of the resected normal colonic mucosa (in vitro) and the rectal mucosa of a healthy volunteer (in vivo) was possible using the endomicroscope. The LCM images for each specimen were compared with the hematoxylin-eosin-stained histopathological cross-sections. Results: The LCM images corresponded well with the conventional hematoxylin-eosin light-microscopic images. The nuclei were not visualized in normal mucosa or hyperplastic polyps. In adenomas with high-grade dysplasia and carcinomas, nuclei were more often visible than in adenomas with low-grade dysplasia. The rate of visualization of nuclei was significantly different (P < 0.01) between these two groups (60.0% vs. 10.3%). In LCM images using endomicroscope, it was possible to recognize the orifices of the colonic glands and goblet cells both in vitro and in vivo. Conclusions: Laser-scanning confocal microscopy provides immediate images that correspond well with those of hematoxylin-eosin staining. An improved probe-type LCM endomicroscope is being developed which should provide better histological images of colorectal lesions in vivo.

Abstract: Previous studies have provided the biological basis for the therapeutic use of enamel matrix derivative (EMD) at sites of periodontal regeneration. A purpose of this study is to determine effects of EMD on cell growth, osteoblastic differentiation and insulin-like growth factor-I (IGF-I) and transforming growth factor-beta 1 (TGF-beta 1) production in human periodontal ligament cells (HPLC). We also examined participation of endogenous IGF-I and TGF-beta 1 with EMD-stimulated cell growth in these cells. HPLCs used in this study were treated with EMD alone or in combination with antihuman IGF-I antibody (anti-hIGF-I) or anti-hTGF-beta 1, recombinant human bone morphogenetic protein-2 (rhBMP-2), 1,25-dihydroxyvitamin D3[1,25(OH)2D3], rhTGF-beta 1 or rhIGF-I. After each treatment, cell growth, the production of IGF-I and TGF-beta 1 and the expression of osteoblastic phenotypes were evaluated. EMD stimulated cell growth in dose-dependent and time-dependent manners. EMD was also stimulated to express IGF-I and TGF-beta 1 at protein and mRNA levels. The EMD-stimulated cell growth was partially suppressed by cotreatment with anti-hIGF-I or anti-hTGF-beta 1, and cell growth was also stimulated by treatment with rhIGF-I or rhTGF-beta 1. rhBMP-2 stimulated alkaline phosphatase (ALPase) activity and ALPase mRNA expression, and 1,25(OH)2D3 stimulated ALPase and osteocalcin mRNA expression. However, EMD showed no effect on the osteoblastic phenotypes expression. These results demonstrated that EMD has no appreciable effect on osteoblastic differentiation, however it stimulates cell growth and IGF-I and TGF-beta 1 production in HPLC, and that these endogenous growth factors partially relate to the EMD-stimulated cell growth in HPLC.

Abstract: The purposes of this study were to investigate the surface morphology, suface roughness of cavities prepared by Er:YAG laser irradiation, and compared the microleakage degree after composite resin restoration with etched bur cavities, in vitro. In each of the 30 human extracted teeth, two shallow cavities were prepared; one prepared with the Er:YAG laser system on the buccal surface, and one produced on the lingual (palatal) surface with a high-speed turbine. Five cavities from each group were investigated by scanning electron microscopy (SEM), and five were subjected to surface roughness analysis by a colour laser three-dimensional (3D) microscope. The remaining cavities were filled with a composite resin and subjected to a microleakage test under thermocycling. Only bur cavities were acid-etched before filling. Statistical analysis was performed using the Mann-Whitney U test; a value of p <0.01 was considered significant. Morphologically, the prepared cavities showed an irregular surface with the absence of a debris-like smear layer; enamel prisms and opening of dentinal tubules were recognised. Surface roughness was significantly increased with the laser system. Microleakage test revealed no significant differences between the laser and bur cavities. Crosscut sections of the cavities with no microleakage showed no gap at the interface. Laser cavity may facilitate good adaptation of composite resin with enamel and dentine, because an increase of surface roughness and the openings of dentinal tubules may facilitate the formation of a hybrid zone, since a primer and an adhesive can penetrate the surface better when the smear layer is removed. It can be concluded that shallow cavity prepared by Er:YAG laser is capable of decreasing microleakage of composite resin restorations, and its efficiency is similar to etched bur cavities.

Abstract: Purpose G-protein-coupled receptors are known to mediate cell growth via divergent signaling pathways. It has been reported that colon cancer cells express muscarinic acetylcholine receptor (mAChR) although their functional role is largely unknown. The aim of this study is to elucidate possible mechanisms responsible for the growth-promoting effect of mAChRs in colon cancer cells by using colon cancer cell line T84.

Abstract: Aims: To test whether 3-week duration recordings of sleep bruxism are correlated with daily behaviors. Methods: Twelve patients with a sleep bruxism disorder were monitored to see if any daily behaviors (stress, physical activity, anger), jaw-pain/headache symptoms, or sleep quality were correlated with their sleep bruxism levels. A telemetric-based system was used for monitoring bruxism levels, which were detected with an intra-appliance piezoelectric film system. Bruxism was defined as a force applied to the occlusal surface of the splint at or above a level of 10% maximum voluntary contraction. Bruxism levels were recorded at night for at least 3 weeks on the 12 subjects in this study (6 females and 6 males). Patients used standard (100 mm) visual analog scaling methods during this period to rate their daily behaviors, sleep quality, and jaw-pain/ headache symptoms in a diary. Correlation analysis was performed between these recorded variables. Results: The subjects demonstrated both bruxism and sleep disturbance, and the mean bruxism score for the male subjects was significantly higher than that for the female subjects. Overall, no single diary variable was consistently correlated with the bruxism levels in these subjects. Conclusion: These data support the conclusion that bruxism is not strongly related to any of the subject's self-monitored daytime activities or sleep quality.