Characterisation of a sucrose-independent in vitro biofilm model of supragingival plaque.

OBJECTIVES: Sugar consumption has been decreasing in Japan, suggesting higher rates of sucrose-independent supragingival plaque formation. For developing an in vitro biofilm model of sucrose-independent supragingival plaque, this study aimed to investigate the compositions and functions on contributing to cariogenicity in comparison with sucrose-dependent biofilm. MATERIALS AND METHODS: An in vitro multispecies biofilm containing Actinomyces naeslundii, Streptococcus gordonii, S. mutans, Veillonella parvula and Fusobacterium nucleatum was formed on 24-well plates in the absence or presence of 1% sucrose. Compositions were assessed by plate culture, scanning electron microscopy and confocal laser scanning microscopy after fluorescent in situ hybridisation or labelling of extracellular polymeric substances (EPS). Functions were assessed by acidogenicity, adherence strength and sensitivities to anticaries agents. RESULTS: Although both biofilms exhibited a Streptococcus predominant bacterial composition, there were differences in bacterial and EPS compositions; in particular, little glucan EPS was observed in sucrose-independent biofilm. Compared with sucrose-dependent biofilm, acidogenicity, adherence strength and antimicrobial resistance of sucrose-independent biofilm were only slightly lower. However, dextranase degradation was substantially lower in sucrose-independent biofilm. CONCLUSION: Our findings suggest that sucrose-independent biofilm may have cariogenicity as with sucrose-dependent biofilm. These in vitro models can help further elucidate plaque-induced caries aetiology and develop new anticaries agents.

Effects of L-ascorbic acid 2-phosphate magnesium salt on the properties of human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: L-Ascorbic acid 2-phosphate magnesium salt (APM) is an L-ascorbic acid (AsA) derivative developed to improve AsA stability and display effective biochemical characteristics. This study aimed to investigate the effects of APM on the functions and properties of human gingival fibroblasts with respect to the prevention of periodontal disease in comparison with those of AsA. MATERIAL AND METHODS: Human gingival fibroblasts were incubated in the presence or absence of APM or L-ascorbic acid sodium salt (AsANa). Intracellular AsA was analysed by HPLC. Collagen synthesis was measured by ELISA and real-time RT-PCR. Intracellular reactive oxygen species (ROS) induced by hydrogen peroxide (H(2)O(2)) were quantified using a fluorescence reagent, and cell damage was estimated with calcein acetoxymethyl ester. Furthermore, intracellular ROS induced by tumor necrosis factor-α (TNF-α) were quantified, and expression of TNF-α-induced interleukin-8 expression, which increases due to inflammatory reactions, was measured by ELISA and real-time RT-PCR. RESULTS: APM remarkably and continuously enhanced intracellular AsA and promoted type 1 collagen synthesis and mRNA expression. Furthermore, APM decreased cell damage through the suppression of H(2)O(2)-induced intracellular ROS and inhibited interleukin-8 production through the suppression of TNF-α-induced intracellular ROS. These effects of APM were superior to those of AsANa. CONCLUSION: These results suggest that APM is more effective than AsANa in terms of intake, collagen synthesis, decreasing cell damage and inhibiting interleukin-8 expression in human gingival fibroblasts. This suggests that local application of APM can help to prevent periodontal disease.

Surgical repair of refractory strictures of esophagogastric anastomoses caused by leakage following esophagectomy.

Refractory strictures of esophagogastric anastomosis caused by leakage following an esophagectomy are a severe complication, for which either repeated balloon dilations or bougies are not necessarily effective. In such a case, surgical repair is quite difficult because the esophageal substitute such as the stomach or colon is usually located in the mediastinum and severely adhesive to the neighboring organs. Furthermore, in case the resected stricture is too long for direct re-anastomosis to be performed, a free jejunal graft or a new esophageal substitute should be prepared. This paper proposes a procedure for the re-reconstruction of refractory stricture in the case of a retrosternal reconstruction with a gastric conduit, which frequently employs pull-up route. The anterior plate of the manubrium was divided medially from the notch to the symphysis with the sternal saw. The manubrium is then removed, bite by bite, like breaking up rocks, with a bone rongeur forceps, starting with the anterior plate, then the posterior plate, from upper median part to the lower and lateral part of the sternum until it reaches the symphysis and the sternoclavicular and the sternocostal joints. It is safer to destroy the manubrium little by little from the anterior side so that the posterior periosteum, which is likely to adhere tightly to the gastric conduit, can be preserved. After the manubrium is almost completely resected and the posterior periosteum of the manubrium is preserved, a median longitudinal incision is carefully made on the periosteum so as not to damage the gastric conduit that may be adhesive to the periosteum. The periosteum was gradually opened bilaterally separating the periostium and the gastric conduit. Although gastroenterological surgeons may hesitate to remove the manubrium, removing the manubrium and preserving the posterior periosteum make it possible to avoid injuring the gastric conduit and to provide a wide view around the stenosis for safely resecting the anastomotic stricture. Furthermore, this procedure allows direct re-anastomosis between the cervical esophagus and the gastric conduit without a complicated reconstruction such as a free jejunal graft. This procedure is strongly recommended as an alternative option so that a second reconstruction can be performed both safely and steadily.

The novel compound NO-1886 increases lipoprotein lipase activity with resulting elevation of high density lipoprotein cholesterol, and long-term administration inhibits atherogenesis in the coronary arteries of rats with experimental atherosclerosis.

We have discovered a novel compound, NO-1886, which possesses a powerful lipoprotein lipase (LPL) activity-increasing action. Administration of NO-1886 increased LPL activity in the postheparin plasma, adipose tissue, and myocardium of rats, and produced a reduction in plasma triglyceride levels with concomitant elevation of HDL cholesterol levels. Administration of NO-1886 increased LPL enzyme mass in postheparin plasma and mRNA activity in epididymal adipose tissue, and it was concluded that the mode of action of this compound is stimulation of tissue LPL synthesis. We also conducted long-term studies to assess the impact of increases in LPL activity and HDL levels on the development of atherosclerotic lesions in rats. Administration of NO-1886 for as long as 90 d significantly decreased the degree of atherosclerotic changes in the coronary arteries of vitamin D2-treated, cholesterol-fed rats. Statistical analysis indicated that increased concentration of HDL is the factor contributing mostly to the prevention of coronary artery sclerosis. In summary, the results of our study indicate that compound NO-1886 increases LPL activity, causing an elevation in HDL levels, and that long-term administration of NO-1886 to rats with experimental atherosclerosis provides significant protection against the development of coronary artery lesions.

Developmental appearance of transcription factors that regulate liver-specific expression of the aldolase B gene.

The region from -202 to -1 of the rat aldolase B (AldB) promoter directs tissue-specific transcription in vitro. Deletion of the half of this promoter distal to its origin reduced its ability for tissue-specific expression. Two protein-binding sites in this distal element have been identified and characterized. One bound a factor named AIF-A, which is probably identical to previously characterized liver-specific factors HNF-1 and APF. The other, containing a CCAAT motif, bound a factor named AIF-B. These two factors were considerably enriched in liver cells compared with other cells not expressing AldB. In the liver, these two factors increased prior to the activation of the AldB gene during development. Liver-specific in vitro transcription assays indicated that the binding of both factors to their target sequences was required for AldB transcription. These results suggest that AIF-A and AIF-B are positively acting transcription factors that regulate tissue-specific and development stage-specific activation of the AldB gene.

2D Thick-section MR digital subtraction angiography for the assessment of dural arteriovenous fistulas.

BACKGROUND AND PURPOSE: Although dynamic contrast-enhanced MR angiography studies for arteriovenous malformations (AVFs) and brain tumors have shown promising results, no formal attempt has yet been made to similarly evaluate dural AVFs. To assess the practical applicability of 2D thick-section contrast enhanced MR digital subtraction angiography (MRDSA) for the diagnosis and management of dural AVFs, MRDSA and intra-arterial digital subtraction angiography (IADSA) were comparatively evaluated. METHODS: We performed 80 consecutive MRDSA studies for 25 dural AVFs, including 11 cavenous sinuses, 9 sigmoid sinuses, 2 tentorial sinuses, one anterior condylar vein, one craniocervical junction, and one spine. MR images were continuously obtained following the initiation of a bolus injection of gadrinium chelates and subtraction images were constructed. We thereafter evaluated the imaging quality and hemodynamic information from all 46 MRDSA images performed in parallel with IADSA in either perioperative or follow-up studies. RESULTS: Most MRDSA images detected early venous filling, sinus occlusion, leptomeningeal venous drainage, and varices. It was difficult, however, to identify the feeding arteries because of both the partial volume effect and a low spatial resolution. Most important, MRDSA accurately detected aggressive lesions with leptomeningeal venous drainage and varices. CONCLUSION: Our MRDSA technique was found to have limited value for depicting all the anatomic details of dural AVFs, though it was able to identify important hemodynamic abnormalities related to the risk of hemorrhaging. MRDSA is therefore useful as a less invasive, dynamic angiographic tool, not only for perioperative studies but also for follow-up studies.

Squamous metaplasia of normal and carcinoma in situ of HPV 16-immortalized human endocervical cells.

The importance of cervical squamous metaplasia and human papillomavirus 16 (HPV 16) infection for cervical carcinoma has been well established. Nearly 87% of the intraepithelial neoplasia of the cervix occur in the transformation zone, which is composed of squamous metaplastic cells with unclear origin. HPV DNA, mostly HPV 16, has been found in 90% of cervical carcinomas, but only limited experimental data are available to discern the role of HPV 16 in this tissue specific oncogenesis. We have initiated in vivo studies of cultured endocervical cells as an experimental model system for development of cervical neoplasia. Using a modified in vivo implantation system, cultured normal endocervical epithelial cells formed epithelium resembling squamous metaplasia, whereas those immortalized by HPV 16 developed into lesions resembling carcinoma in situ. In contrast, their ectocervical counterparts formed well differentiated stratified squamous epithelium and a lesion with mild dysplastic change, respectively. The HPV 16-immortalized cells showed in vivo cytokeratin expression patterns similar to their respective normal counterparts, confirming their different origins. Thus, this study provides direct experimental evidence for the transformation of simple epithelial cells of endocervical origin into stratified squamous metaplasia and indicates the differential susceptibility of endo- and ectocervical epithelial cells for conversion to cancer by HPV 16.

Luseogliflozin, A Sodium Glucose Co-transporter 2 Inhibitor, Alleviates Hepatic Impairment in Japanese Patients with Type 2 Diabetes.

Luseogliflozin, a selective inhibitor of sodium glucose co-transporter 2 (SGLT2), was previously shown to improve the blood glucose and hemoglobin A1c (HbA1c) levels of patients with type 2 diabetes in a clinical setting. Although patients with type 2 diabetes often have hepatic impairment, few reports have been published concerning the influence of luseogliflozin on HbA1c and hepatic function in patients with type 2 diabetes accompanied by hepatic impairment. The present study was undertaken to evaluate the influence of luseogliflozin on HbA1c and hepatic function in patients with type 2 diabetes divided into 2 groups according to hepatic function parameters (a normal group and an elevated group). In this study, luseogliflozin significantly improved both HbA1c and body weight to similar extents in both the normal group and the elevated group, accompanied by marked reductions in the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (γ-GTP) levels. These results suggested that luseogliflozin can be safely used in patients with type 2 diabetes who also exhibit hepatic impairment. The results additionally suggest the possibility that luseogliflozin might be capable of alleviating hepatic impairment in patients with type 2 diabetes.

The Beneficial Effects of the DPP-4 Inhibitor Alogliptin on Hemoglobin A1c and Serum Lipids in Japanese Patients with Type 2 Diabetes.

It has been reported that dipeptidyl peptidase-4 (DPP-4) inhibitors improve hemoglobin A1c (HbA1c) levels in diabetic patients and may also improve the serum lipids. However, few studies have examined relationship between the effects of the DPP-4 inhibitor and the pretreatment HbA1c levels in diabetic patients. Furthermore, it has been reported that prolonged treatment with DPP-4 inhibitors may make glycemic control difficult in some patients. In the present study, we investigated (1) the effect of the DPP-4 inhibitor alogliptin on HbA1c, blood glucose (BG), and serum lipid in Japanese patients with type 2 diabetes, (2) the relationship between the HbA1c levels at baseline and the effects of alogliptin, and (3) the effects of switching of the DPP-4 inhibitor to alogliptin after 12 months' administration of sitagliptin on glycemic control and serum lipids. After 6-months' treatment with alogliptin, we found reductions of HbA1c, BG, and serum total cholesterol, and LDL cholesterol levels. Pretreatment level of HbA1c was well correlated with the degree of reduction of both HbA1c and BG levels after the treatment. Also, alogliptin kept levels of HbA1c and BG reduced by sitagliptin for 12 months, and relapsing of these levels and serum lipids were not observed. This study revealed that alogliptin improved HbA1c, BG, and serum lipid profiles in type 2 diabetic patients, and the effect of alogliptin on HbA1c and BG levels was correlated with HbA1c level at pretreatment. Furthermore, long-term treatment with alogliptin did not cause relapsing of glycemic control and serum lipids.

Ubiquitous factors that interact simultaneously with two distinct cis-elements on the rat aldolase B gene promoter.

The proximal promoter region (nucleotide -202 to -1) of the rat aldolase B gene confers liver-specific transcription, and contains three indispensable protein-binding sites (site A, site B and site C). Site A binds HNF-1 and HNF-3, and site B binds NF-Y and a CCAAT-binding factor AlF-B (Tsutsumi et al. (1989) Mol. Cell. Biol. 9, 4923-4931; Raymondjean et al. (1991) Nucleic Acids Res. 19, 6145-6153), trans-acting factors that interact with site C, however, have not been well characterized. In this study, we identified specific factors that bind site C by two-dimensional gel electrophoretic analyses. The factors interacted with two distinct cis-elements; site C and site B. This observation, together with the fact that site C contains a C/EBP binding motif, implied that the site C-binding factors are members of C/EBP family. However, analyses of their binding characteristics, their relative molecular masses, and their distribution in different cell types showed that the site C binding factors are different from known members of C/EBP family.

Involvement of inorganic polyphosphate in expression of SOS genes.

Inorganic polyphosphate (poly(P)) is a linear polymer that has been found in every organism so far examined. To elucidate the functions of poly(P) in the regulation of gene expression, the level of cellular poly(P) in Escherichia coli was reduced to a barely detectable concentration by overproduction of exopolyphosphatase (exopoly(P)ase) with a plasmid encoding yeast exopoly(P)ase (Shiba et al., Proc. Natl. Acad. Sci. USA 94 (1997) 11210-11215). It was found that exopoly(P)ase-overproducing cells were more sensitive to UV or mitomycin C (MMC) than were control cells. Poly(P) accumulation was observed after treatment with MMC, whereas the poly(P) level was below the detectable level in cells that overproduced exopoly(P)ase. When exopoly(P)ase-overproducing cells were transformed again by a multiple copy number plasmid that carries the polyphosphate kinase gene (ppk), the cells accumulated a great amount of poly(P) and restored the UV and MMC sensitivities to the level of control cells. In exopoly(P)ase-overproducing cells, the expression of recA and umuDC were not induced by MMC. In addition, a strain containing multiple copies of ppk accumulated not only a large amount of poly(P) but also recA mRNA. Since recA expression was induced in a recA-deletion strain harboring a plasmid with the ppk gene, poly(P) could be necessary for regulating the expression of SOS genes without depending on the RecA-LexA regulatory network.

A novel variant of translation elongation factor-1beta: isolation and characterization of the rice gene encoding EF-1beta2.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Prognostic factors in adenocarcinoma of the gastric cardia: pathologic stage analysis and multivariate regression analysis.

PURPOSE: To clarify the pathologic stages of adenocarcinoma of the gastric cardia in which the prognosis is worse than in adenocarcinoma of the middle or distal part of the stomach, and to determine prognostic factors in these stages by multivariate analysis. PATIENTS AND METHODS: We analyzed 2,536 cases of surgically resected gastric adenocarcinoma of all pathologic stages. Four hundred seventy-two cases of gastric carcinoma, in which cumulative survival of gastric cardia was poor, were subjected to Cox regression analysis for prognostic factors, and to logistic regression analysis for factors influencing venous or lymphatic invasion. RESULTS: The prognosis of adenocarcinoma of the gastric cardia was inferior when compared with similarly staged carcinomas of the middle or lower part of the stomach when there was invasion of proper muscle layer or subserosal layer, with no lymph node metastasis or with only adjacent (group 1) lymph nodes metastases (T2N0 or T2N1, according to the Japanese classification). In these stages, the prognostic factors were age, histologic type, venous invasion, and location of the tumor in the upper part of the stomach. Tumor location in the upper stomach was also a predictor for the presence of venous invasion. CONCLUSION: The prognosis of adenocarcinoma of the gastric cardia is poor in patients with T2 tumors with no or few lymph node metastases. Additional treatment after surgery may be necessary to improve the survival of this population.

Correction of hypertriglyceridemia with low high-density lipoprotein cholesterol by the novel compound NO-1886, a lipoprotein lipase-promoting agent, in STZ-induced diabetic rats.

We have previously reported that the novel compound NO-1886 increased lipoprotein lipase (LPL) activity, with resulting elevation of high-density lipoprotein (HDL) cholesterol in normal rats (J Clin Invest 92:411-417, 1993). The aim of this study was to ascertain whether the compound has the same action in diabetes, because hypertriglyceridemia with low HDL cholesterol is an extremely common concomitant condition in diabetes. Streptozotocin-induced diabetic rats showed marked elevation of plasma triglyceride and reduction of HDL cholesterol. Both single and repeated administration of NO-1886 increased postheparin plasma LPL activity, with resulting reduction of plasma triglyceride and elevation of HDL cholesterol. Repeated administration increased the amount of LPL mRNA in adipose tissue and myocardium. The compound had no effects on plasma glucose and insulin levels. Our study indicates that the compound is potentially beneficial for the treatment of hypertriglyceridemia with low HDL cholesterol in diabetes.

Structure and genomic organization of the rat aldolase B gene.

The structure of the chromosomal gene encoding rat aldolase isozyme B has been elucidated by sequence analysis of cloned genomic DNA. This gene comprises about 14 X 10(3) base-pairs of DNA, and is separated into nine exons by eight intervening sequences. A presumed transcription-initiation site was assigned by S1 nuclease protection mapping, and T-A-T-A and C-C-A-A-T boxes were found to be 25 and 126 base-pairs, respectively, upstream from this initiation site. There are three characteristic sequences of 100 to 200 base-pairs within the region of 870 base-pairs flanking the 5' side of the gene. These sequences are flanked on either side by direct repeats and terminate with an A-rich stretch of nucleotides. One of them has block homology with a region in an "ID sequence", which is reported to be an element for tissue-specific gene regulation and differentiation. The other two are analogous at the sequence organizational level with a sort of dispersed repeat, the "Alu family". These features suggest that these regions are involved in gene regulation and, also, imply evolutionary events such as duplication or insertion. Comparison of this gene sequence with the rabbit aldolase A complementary DNA sequence revealed some bias in the frequency of nucleotide replacement among the exons, suggesting selective evolutionary conservation of particular exons encoding functional domains. Comparison with the human aldolase B complementary DNA sequence revealed no such tendency; the homology between the two sequences was very high (about 89%), and nucleotide replacements were randomly distributed throughout the protein-coding region.

[Successful removal of T4 lung cancer with left atrial invasion after induction chemotherapy].

A 53-year-old man presented with cough, sputa and chest pain. The chest X-ray revealed a large mass shadow in the right lower lobe. Massive tumor extending into the left atrium was diagnosed by computed tomography (CT). The brushing cytology by broncoscopy was squamous cell carcinoma and its stage was IIIB. Chemothrapy using cisplatin, paclitaxel and gemcitabine hydrochloride was performed 8 courses during 6 months. The effect of the chemotherapy was complete response, enabling the surgical treatment. The right pneumonectomy with partial resection of the left atrium was performed by using vascular clamp. The defect of the left atrium could be sutured directly. Wide-spread necrotic change with very small amount of cancer cells in the atrial wall was confirmed by pathology. The patient has been well for 3 years and 6 months after surgery.

Profiles of embryonic nuclear protein binding to the proximal promoter region of the soybean ß-conglycinin α subunit gene.

ß-Conglycinin, a major component of seed storage protein in soybean, comprises three subunits: α, α' and ß. The expression of genes for these subunits is strictly controlled during embryogenesis. The proximal promoter region up to 245 bp upstream of the transcription start site of the α subunit gene sufficiently confers spatial and temporal control of transcription in embryos. Here, the binding profile of nuclear proteins in the proximal promoter region of the α subunit gene was analysed. DNase I footprinting analysis indicated binding of proteins to the RY element and DNA regions including box I, a region conserved in cognate gene promoters. An electrophoretic mobility shift assay (EMSA) using different portions of box I as a probe revealed that multiple portions of box I bind to nuclear proteins. In addition, an EMSA using nuclear proteins extracted from embryos at different developmental stages indicated that the levels of major DNA-protein complexes on box I increased during embryo maturation. These results are consistent with the notion that box I is important for the transcriptional control of seed storage protein genes. Furthermore, the present data suggest that nuclear proteins bind to novel motifs in box I including 5'-TCAATT-3' rather than to predicted cis-regulatory elements.

DPP-4 Inhibitor Teneligliptin Improves Insulin Resistance and Serum Lipid Profile in Japanese Patients with Type 2 Diabetes.

Dipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to improve the glycemic control and blood hemoglobin A1c (HbA1c) concentrations. However, there are few reports as yet suggesting that DPP-4 inhibitors may also improve insulin resistance and the serum lipid profile in the clinical setting. This study was aimed at investigating the effect of 14-week treatment with teneligliptin (20 mg/day) on the homeostasis model assessment ratio (HOMA-R), an indicator of insulin resistance, and serum lipid profile in 9 patients with type 2 diabetes. The treatment produced a significant decrease of the blood glucose and HbA1c concentration (blood glucose: p=0.008; HbA1c: p=0.038), and also improved HOMA-R (p=0.039). Furthermore, the patients showed elevation of the serum HDL-cholesterol level (p=0.032), and a tendency towards reduction of the serum triglyceride level. The results indicate that teneligliptin acts not only to improve the blood glucose control, but also to improve the insulin resistance and serum lipid profile in Japanese type 2 diabetes patients.

Jaw movement-related primary somatosensory cortical area in the rat.

It has anatomically been revealed that the rostral part of the rat primary somatosensory cortex (S1) directly projects to the dorsal part of the trigeminal oral subnucleus (dorVo) and the dorsal part of juxtatrigeminal region (dorVjuxt), and that the dorVo and dorVjuxt contain premotoneurons projecting directly to the jaw-opening or jaw-closing motoneurons in the trigeminal motor nucleus (Vmo). However, little is known about how the rostral S1 regulates jaw movements in relation to its corticofugal projections. To address this issue, we performed intracortical microstimulation of the rat rostral S1 by monitoring jaw movements and electromyographic (EMG) activities. We for the first time found that low-frequency long-train stimulation of the rostral S1 induced single sustained opening of the jaw with elevated EMG activities of the anterior digastric muscles (jaw-opener). The effective sites for the low-frequency long-train stimulation overlapped the S1 sites where traditional high-frequency short-train stimulation was effective to induce single twitch-like jaw movement. We also found that the effective sites for the two kinds of train stimuli were included in the rostral S1 area, which has previously been identified to send direct projections to the dorVo or the dorVjuxt. Specifically, the most effective stimulation sites for the two kinds of train stimuli were located in the rostralmost part of S1 which has been reported to emanate strong direct projections to the dorVjuxt but less to the dorVo. Therefore, the present study suggests that the rat rostral S1, especially its rostralmost part, plays an important role in controlling jaw movements by activation of direct descending projections from the rostral S1 to the trigeminal premotoneuron pools, especially to the dorVjuxt.