Phenytoin-induced gingival overgrowth caused by death receptor pathway malfunction.

OBJECTIVE: In this study, we investigated the role of phenytoin (PHT) in death receptor-induced apoptosis of gingival fibroblasts to clarify the mechanism of PHT-induced gingival overgrowth. METHODS: Human gingival fibroblasts were cultured to semiconfluence and treated with PHT (0.025, 0.1, 0.25, and 1.0 µM) for 48 h, and then, the apoptotic cell numbers were relatively determined by absorptiometry. After 24 h of 0.25 µM PHT treatment, caspase activity was measured by absorptiometry, apoptotic and cell cycle phase distribution was analyzed by flow cytometry, expression levels of apoptotic genes were quantified by real-time qPCR, and expression of apoptotic proteins was detected by Western blot analysis. After 48 h of 0.25 µM PHT treatment, appearance of apoptotic cells was detected by TUNEL assay. RESULTS: PHT treatment decreased the proportion of apoptotic cells in gingival fibroblasts compared to a serum-free control culture in response to the protein changes as follows: PHT upregulated c-FLIP and, in turn, downregulated FADD, caspase-8, and caspase-3; PHT upregulated c-IAP2 and downregulated TRAF2; PHT downregulated caspase-9 and caspase-3 via decreased RIPK1 activity and increased Bcl-2 activity. CONCLUSION: PHT-induced gingival overgrowth may result from the above-mentioned mechanisms involving apoptosis inhibition in gingival fibroblasts.

A compact low-temperature hydrogen ion beam apparatus for in situ physical property measurements.

A new compact low-temperature hydrogen ion beam apparatus has been developed for in situ physical property measurements. Introduction of hydrogen can significantly alter the physical properties of materials. Conventional methods such as exposure to H2 gas are limited to materials having hydrogen sorption. The present method is, in principle, applicable to any material of interest. Our setup provides a facile way to conduct both low-temperature hydrogen ion beam irradiation and in situ electrical resistivity measurements, which enables observation of novel physical properties induced by the low-temperature irradiation. The lowest temperature of 3.8 K was achieved by utilizing a newly designed rotatable radiation shield and a closed-cycle cryostat, which is advantageous for long-time low-temperature experiments for heavy hydrogen doping and in situ analysis. It was found that the resistivity of ZnO largely decreased by hydrogen ion beam irradiation at 50 K. Furthermore, the in situ measurements revealed an unforeseen irreversible thermal hysteresis for resistivity.

Effects of DSP-8658, a novel selective peroxisome proliferator-activated receptors a/γ modulator, on adipogenesis and glucose metabolism in diabetic obese mice.

AIMS/INTRODUCTION: Peroxisome proliferator-activated receptors (PPARs) play a key regulating role in homeostasis. In this study, we investigated the effects of DSP-8658, a novel selective PPARa/γ modulator, on adipogenesis and glucose metabolism in diabetic obese mice and compared these effects to those of pioglitazone, a PPARγ full agonist. MATERIALS AND METHODS: DSP-8658 functional activity was assessed by PPARγ-target genes expression in adipose 3T3-L1 cells and its anti-diabetic efficacy evaluated in db/db mice. The effects of DSP-8658 on adipogenesis were investigated diet induced obese (DIO) KK-A(y) mice. RESULTS: DSP-8658 reduced the expression of PPARγ-target gene 11 beta hydroxysteroid dehydrogenase type 1 with an EC50 value 2.1-fold that of pioglitazone and 28.4-fold that of rosiglitazone. On the other hand, DSP-8658 increased the expression of fatty acid binding protein 4 and glycerol kinase genes with EC50 values 33-fold and >15-fold those of pioglitazone and 163-fold and >38-fold those of rosiglitazone, respectively. In db/db mice, DSP-8658, like pioglitazone, decreased blood glucose, HbA1c, and plasma triglyceride levels and increased plasma insulin concentration and pancreatic insulin contents. In DIO KK-A(y) mice, DSP-8658, unlike pioglitazone, decreased subcutaneous adipose tissue weight and mean adipocyte size. However, both DSP-8658 and pioglitazone improved blood glucose and HbA1c levels with similar efficacy. Although DSP-8658 did not change the expression levels of fatty acid transport protein 1 and glycerol kinase genes in subcutaneous adipose tissue of KK-A(y) mice, pioglitazone increased these gene expression levels. CONCLUSION: Unlike PPARγ full agonists, DSP-8658 ameliorates blood glucose without increasing adipogenesis in diabetic obesity mice.

Hemokinin-1 mediates pruriceptive processing in the rat spinal cord.

Hemokinin-1 (HK-1) is a new mammalian tachykinin peptide consisting of the amino acid sequence similar to substance P (SP). Although the function of SP, a representative tachykinin peptide, has been well established in the pain system, that of HK-1 has not yet been elucidated. [Leu(11)]-SP had an antagonistic effect on SP-induced scratching behavior, suggesting that [Leu(11)]-HK-1 may also attenuate the induction of scratching behavior by HK-1. Thus, the effects of a pretreatment with [Leu(11)]-HK-1 were evaluated to clarify the function of HK-1. The intrathecal administration of [Leu(11)]-HK-1 attenuated the induction of scratching by HK-1, but not SP, while [Leu(11)]-SP reduced the induction of scratching by SP, but not HK-1. These results indicated that [Leu(11)]-HK-1 may be a more specific antagonist of HK-1-preferred receptors and [Leu(11)]-SP has an antagonistic effect on the SP-preferred receptor, the neurokinin 1 receptor. In the formalin test for examining noxious response, the intrathecal administration of [Leu(11)]-SP, but not [Leu(11)]-HK-1, reduced the number of flinchings and c-Fos-positive cells in the spinal dorsal horn following formalin injection into the plantar region of the hind paw. These results indicated that SP, but not HK-1, is involved in nociceptive processing at the spinal level. To evaluate the involvement of HK-1 and SP in pruritic processing, the effect of [Leu(11)]-HK-1 and [Leu(11)]-SP on the induction of scratching behavior and c-Fos expression by serotonin (5-HT) and histamine was evaluated. The increased induction of scratching behavior and c-Fos expression by 5-HT and histamine was markedly attenuated by pretreatment with both [Leu(11)]-HK-1 and [Leu(11)]-SP, suggesting that HK-1 and SP may be involved in pruritic processing. These results indicate that HK-1 is involved in pruritic processing and [Leu(11)]-HK-1 is a valuable tool for clarifying the mechanisms underlying pruritic processing.

An amino-terminal fragment of hemokinin-1 has an inhibitory effect on pruritic processing in rats.

Hemokinin-1 (HK-1) is a peptide encoded by the preprotachykinin gene, TAC-4, and shares the hydrophobic carboxyl-terminal (C-terminal) region common to mammalian tachykinin peptides, such as substance P (SP). It is generally believed that C-terminal fragments of SP elicit an excitatory effect, while pretreatment with amino-terminal (N-terminal) fragments of SP inhibits the function of SP; however, there is no available information on HK-1. Therefore, to clarify the characteristics of C-terminal and N-terminal fragments of HK-1, HK-1 was divided into HK-1 (1-5) as the N-terminal fragment and HK-1 (6-11) as the C-terminal fragment based on the similarity of amino acids between HK-1 and SP. Intrathecal administration of HK-1 (6-11) induced scratching behavior similar to HK-1, while HK-1 (1-5) hardly induced scratching. Pretreatment with HK-1 (1-5), however, attenuated scratching induced by HK-1 and SP, whereas pretreatment with SP (1-5) attenuated SP-induced scratching, but not HK-1. Furthermore, intrathecal administration of HK-1 (1-5) and SP (1-5) markedly attenuated the induction of flinching and enhancement of c-Fos expression in the spinal cord following the intradermal administration of formalin, a noxious stimulant, while pretreatment with HK-1 (1-5), but not SP (1-5), markedly attenuated the induction of scratching behavior by subcutaneous administration of pruritic agents, such as serotonin or histamine. Taken together, these findings indicate that HK-1 (1-5) suppresses pruritic and nociceptive processing, while SP (1-5) suppresses nociceptive processing. Therefore, it is suggested that HK-1 (1-5) may be a useful tool for revealing pruritic processing and HK-1 may play a crucial role in pruritic processing.

c-MYC overexpression with loss of Ink4a/Arf transforms bone marrow stromal cells into osteosarcoma accompanied by loss of adipogenesis.

The development of cancer is due to the growth and proliferation of transformed normal cells. Recent evidence suggests that the nature of oncogenic stress and the state of the cell of origin critically affect both tumorigenic activity and tumor histological type. However, this mechanistic relationship in mesenchymal tumors is currently largely unexplored. To clarify these issues, we established a mouse osteosarcoma (OS) model through overexpression of c-MYC in bone marrow stromal cells (BMSCs) derived from Ink4a/Arf (-/-) mice. Single-cell cloning revealed that c-MYC-expressing BMSCs are composed of two distinctly different clones: highly tumorigenic cells, similar to bipotent-committed osteochondral progenitor cells, and low-tumorigenic tripotent cells, similar to mesenchymal stem cells (MSCs). It is noteworthy that both bipotent and tripotent cells were capable of generating histologically similar, lethal OS, suggesting that both committed progenitor cells and MSCs can become OS cells of origin. Shifting mesenchymal differentiation by depleting PPARγ in tripotent MSC-like cells and overexpressing PPARγ in bipotent cells affected cell proliferation and tumorigenic activity. Our findings indicate that differentiation potential has a key role in OS tumorigenic activity, and that the suppression of adipogenic ability is a critical factor for the development of OS.

An improved computer-aided diagnosis scheme using the nearest neighbor criterion for determining histological classification of clustered microcalcifications.

OBJECTIVES: Our purpose was to evaluate the potential usefulness of the nearest neighbor case which was assumed to be the similar case in a CAD scheme for determining the histological classification of clustered microcalcifications. METHODS: Our database consisted of current and previous magnification mammograms obtained from 93 patients before and after three-month follow-up examination. It included 11 invasive carcinomas, 19 noninvasive carcinomas of the comedo type, 25 non-invasive carcinomas of the noncomedo type, 23 mastopathies, and 15 fibroadenomas. Six objective features on clustered microcalcifications were first extracted from each of the current and the previous images. The nearest neighbor case was then identified by the Euclidean distance in the previous and current feature-space. The histological classification of an unknown new case in question was assumed to be the same as that of the nearest neighbor case which has the shortest Euclidean distance in our database. RESULTS: The classification accuracies were 90.9% for invasive carcinoma, 89.5% for noninvasive carcinoma of the comedo type, 96.0% for noninvasive carcinoma of the noncomedo type, 82.6% for mastopathy, and 93.3% for fibroadenoma. These results were substantially higher than those with our previous CAD scheme. CONCLUSION: The nearest neighbor criterion was useful in a CAD scheme for determining the histological classification.

Dantrolene ameliorates delayed cell death and concomitant DNA fragmentation in the rat hippocampal CA1 neurons subjected to mild ischemia.

The present study investigated whether dantrolene, which inhibits the Ca(2+) release from the intracellular Ca(2+) store sites, reduced nuclear DNA fragmentation and produced neuronal protection in a model of global forebrain ischemia. Male Wistar rats were subjected to four-vessel occlusion (4VO) for 5 min and then infused continuously with dantrolene or vehicle into the cerebral ventricle for 3 days. The intact rats did not undergo any intervention. The number of viable and DNA nick-end-labeled neurons in the hippocampal CA1 were evaluated 4 days after the ischemia. The number of viable neurons in the dantrolene-treated rats was significantly higher than that in the vehicle-treated rats and lower than that in the intact animals (P<0.01 and <0.05, respectively). The number of DNA nick-end-labeled nuclei was significantly lower in dantrolene-treated rats compared with the vehicle-treated animals (P<0.0001). No nick-end labeling was observed in the intact animals. A linear correlation was found between the number of viable cells and nick-end labeled nuclei in the CA1 (r=0.91, P<0.0001). These results suggest that the postischemic intraventricular dantrolene is effective in precluding neuronal death and concomitant nuclear DNA fragmentation following transient global ischemia.

p48 subunit of mouse PTF1 binds to RBP-Jkappa/CBF-1, the intracellular mediator of Notch signalling, and is expressed in the neural tube of early stage embryos.

BACKGROUND: Development of the pancreas and the nervous tissues is regulated by common transcription factors. A basic helix-loop-helix protein, p48 of pancreas transcription factor 1 (PTF1), is essential for differentiation of the exocrine acinar cells. RESULTS: We isolated PTF1 p48 from 9.5-day mouse embryos as a binding protein of RBP-Jkappa, a mediator of Notch signalling. p48 bound to RBP-Jkappa more strongly than and in a distinct way from Notch1. In 9.5-12.5 day embryos, p48 was expressed in the dorsal part of the neural tube as well as in the pancreatic buds. Two lines of evidence suggested functions of p48 in neurogenesis: (i) expression of p48 was induced in P19 cells when they committed to neural fate upon retinoic acid treatment, and (ii) p48 over-expressed in Xenopus embryos repressed the development of neuronal precursors. p48 inhibited the MASH1-activated transcription from the E-box, while p48 stimulated transcription from the PTF1 motif synergistically with E47. The p48/E47-activated transcription from the PTF1 motif was stimulated further by RBP-Jkappa and RBP-Jkappa derivatives that mimicked the active RBP-Jkappa/Notch complex. CONCLUSIONS: In developing embryos, p48 is expressed in both the nervous system and the pancreas. p48 inhibits neuronal differentiation. We propose possible mechanisms for this inhibition.

Intracerebroventricular propofol is neuroprotective against transient global ischemia in rats: extracellular glutamate level is not a major determinant.

Excessive glutamate accumulation in extracellular space due to ischemia in the central nervous system (CNS) is believed to initiate the cascade toward irreversible neuronal damage. An intravenous general anesthetic, propofol (2,6-diisopropylphenol) has been implicated to be neuroprotective against cerebral ischemia. The purpose of this study was to test the hypothesis that intracerebroventricular propofol produced a reduction in extracellular glutamate level during global ischemia and the resultant neuroprotection. Adult male Wistar rats were anesthetized with halothane in nitrous oxide/oxygen and mechanically ventilated. Propofol (3 or 10 mg/kg), Intralipid((R)) as a vehicle for propofol, or artificial cerebrospinal fluid (aCSF) was administered into the cerebral ventricles 15 min prior to a 10-min forebrain ischemia elicited by the four-vessel occlusion. Extracellular glutamate concentration in the hippocampal CA1 was continuously monitored during the peri-ischemic period with a microdialysis biosensor. Neuronal cell loss in the hippocampal CA1 was evaluated by cresyl-violet staining of sections 7 days later. Propofol (3 and 10 mg/kg) and Intralipid, compared with aCSF, similarly reduced the extracellular glutamate accumulation during the peri-ischemic period (P<0.05), indicating that the extracellular glutamate reduction that was seen primarily reflects the effect of Intralipid. The number of intact neurons in the hippocampal CA1 in propofol 10 mg/kg-treated rats was significantly higher than that in rats treated with propofol 3 mg/kg, Intralipid, or aCSF (P<0.05). We conclude that intracerebroventricular propofol exhibits neuroprotection against transient global forebrain ischemia; however, the extracellular glutamate level during ischemia is not a major determinant of this neuroprotection.

'Tako-Tsubo' transient ventricular dysfunction: a case report.

During admission for investigation of dysphagia, an 82-year-old woman suddenly complained of dyspnea, which was followed by cardiogenic shock. Her symptoms, electrocardiogram, echocardiogram and laboratory data were compatible with an extensive acute anterior myocardial infarction. Emergency cardiac catheterization showed no atheromatous narrowing in any coronary artery. However, the contractions of the left and right ventricles were diffusely and severely impaired, except for some hyperkinesis of the basal area. The asynergy, as well as the abnormalities on the ECG, improved almost to normal by the 35th hospital day. An endomyocardial biopsy from the right ventricle during the acute phase showed atypical myocardial damage with proliferation of fine collagen fibers and small round-cell infiltration including polymorphologic leukocytes. This type of transient cardiac disorder has recently been described in Japan, and is called 'Tako-tsubo cardiomyopathy' because of the characteristic appearance of the left ventricular asynergy. In the present case, ventricular asynergy was not limited to the left ventricle, but was also present in the right ventricle.

[Accidental pyriform sinus perforation with Savary-Gilliard esophageal bougie during general anesthesia].

A 71-year-old female with esophagohiatal hernia underwent a laparoscopic Nissen's fundoplication under general anesthesia. During the operation a Savary-Gilliard bougie was temporarily inserted to the esophagus to prevent esophagostenosis. After the emergence from anesthesia, the patient complained of severe back pain and developed tachypnea and a low SpO2 associated with an enlargement of mediastinal shadow on the chest X-ray, suggesting mediastinal perforation. Examination by endoscopy and thoracotomy revealed a pyriform sinus perforation reaching down to the mediastinum. This case demonstrates the possibility of accidental perforation by various devices inserted blindly to esophagus, especially during general anesthesia, and the importance of close observation of patients after the emergence from anesthesia.

[Calcification in culprit lesions of coronary artery disease].

Coronary calcification, a type of coronary atherosclerosis, has recently been closely examined in clinical cardiology because its presence may influence the selection of interventional therapy. In addition, plaque instability is one of the most important factors in the mechanism of acute coronary syndrome, and calcium deposit is frequently detected in advanced lesions. However, little is known about the clinical significance of coronary calcification. The incidence of calcium deposits was investigated in the culprit lesions (culprit coronary calcification) of patients with serious coronary artery disease to discover any cardioprotective effect. Initial coronary angiography was performed in 179 consecutive patients with acute myocardial infarction with Q wave on electrocardiography (AMI group; male 139, female 40, mean age 60.2 +/- 10 yr) and in 119 consecutive patients with stable effort angina pectoris (SAP group; male 78, female 41, mean age 63.8 +/- 8 yr) for which balloon plasty or bypass surgery was necessary from 1990 to 1997. Culprit coronary calcification was defined positive if the calcification deposit was present cinefluoroscopically within 5 mm from the culprit point. The culprit point was defined as the narrowest point after successful intracoronary thrombolytic therapy or the latest point to be dilated during a balloon inflation in direct or rescue percutaneous transluminal coronary angioplasty in the AMI group, and the narrowest point of the culprit lesion in the SAP group. There was no statistical difference in clinical background between the 2 groups other than male dominance in the AMI group and high incidence of family history of ischemic heart disease in the SAP group (p < 0.05). Culprit coronary calcification in patients over 50 years old was less frequently positive in the AMI group than the SAP group (26% vs 66%, p < 0.005, respectively). In younger patients under 50 years old, the incidence of culprit coronary calcification was low (14-15%) in both groups. Culprit coronary calcification was frequently positive in the right or the left anterior descending coronary artery in the SAP group (p < 0.005). There was no incidental sex difference of culprit coronary calcification. This comparison suggests that if a plaque contains cinefluoroscopically visible calcification, it may be regarded as less vulnerable or having a history of chronic process of atherosclerosis which results in protecting plaque rupture.

Localization of the delta subunit in the Escherichia coli F(1)F(0)-ATPsynthase by immuno electron microscopy: the delta subunit binds on top of the F(1).

The binding site of the delta subunit in the F(1)F(0)-ATPsynthase from Escherichia coli has been determined by electron microscopy of negatively stained, antibody-decorated enzyme molecules. The images show that the antibody is bound at the very top of the F(1) domain indicating that at least part of delta is bound in the dimple formed by the N termini of the alpha and beta subunits. The data may explain why there is only one binding site for delta on the F(1) despite there being three identical alphabeta pairs. The finding also implies that the b subunits of the F(0) have to extend all the way from the membrane surface to the very top of the F(1) domain to make contact with the delta subunit.

Protective effect of nisoldipine on myocardial ischemia during coronary bypass surgery.

BACKGROUND: Nisoldipine, a calcium antagonist, was assessed for myocardial protection and the prevention of reperfusion injury in patients undergoing CABG. METHODS: Of the 34 subjects undergoing CABG in this study, 20 were given nisoldipine orally at 10 mg/day for 2 weeks before surgery (N group) and the other 14 untreated controls (C group). Myocardial protection was conducted via ante-grade cold blood cardioplegia at 20-minute intervals. RESULTS: Myocardial blood flow was significantly higher in the N group (67.8 +/- 21.8 ml/100 g vs. 47.2 +/- 14.4 ml/100 g, p < 0.05) after cardiopulmonary bypass. Serum interleukin-6 levels were significantly lower in the N group 1 hour after reperfusion (116 +/- 58 vs. 409 +/- 362 pg/ml, p < 0.05), as were serum lactate dehydrogenase levels immediately after surgery (888 +/- 268 vs. 1350 +/- 486 IU/L, p < 0.05). The N Group showed a better left ventricle stroke work index 6 hours after surgery (43 +/- 8 vs. 36 +/- 9 g.m/m2). Dopamine dosage in the N group on postoperative day 1 was lower than in controls (5.3 +/- 1.9 vs. 3.0 +/- 2.4 micrograms/kg/min). CONCLUSIONS: Preoperative nisoldipine treatment increased blood flow in the postischemic myocardium and prevented myocardial damage and reperfusion injury to some extent.

Defective binding of IRFs to the initiator element of interleukin-1beta-converting enzyme (ICE) promoter in an interferon-resistant Daudi subline.

To investigate mechanisms of interferon (IFN) resistance, we have established an IFN-resistant Daudi subline (Daudi(res)), which is 1 X 10(4) times more resistant to IFN-alpha than parental cells. Among the IFN-inducible genes examined, only ICE mRNA expression was deficient in Daudi(res) cells. We then analyzed the regulatory mechanisms of ICE transcription, and found that IFN-induced activation of the ICE promoter was dependent on the binding of IRFs to its initiator (Inr) element. Inr binding of IRFs was markedly diminished in Daudi(res) cells, and forced expression of IRF-1 was able to activate the ICE promoter to the level of parental cells. These results suggest that IRFs and their target genes, as represented by ICE in this study, are involved in IFN resistance.

Intrafamilial distribution of mutans streptococci in Japanese families and possibility of father-to-child transmission.

The purpose of this study was to investigate the intrafamilial distribution of mutans streptococci in Japanese families using chromosomal DNA fingerprinting with three endonucleases; EcoRI, HindIII and HaeIII. The analysis of 1,908 isolates cultured from the dental plaque of 76 subjects from 20 families (20 married couples and 36 of their children) resulted in the identification of 144 genotypes containing 114 strains of Streptococcus mutans (serotype c, 66.7%; e, 12.5%) and 30 strains of S. sobrinus (d, 13.2%; g, 7.6%). A mean of 1.89 genotypes (from one to four) was harbored in individual subjects, and a mean of 4.10 genotypes from two to seven was harbored in individual families. Among the 70 genotypes found in the children, 36 (51.4%) were in agreement with their mothers and 22 (31.4%) were in agreement with their fathers. The other genotypes (18.6%) did not correspond with the parents. Homologous strains between parents were found in only two couples. This result showed that fathers or others as well as mothers can be sources of transmission. Further, the serotype d, e and g strains showed significantly higher probabilities of transmission than serotype c.

[Tracheal obstruction caused by bucking during general anesthesia in a patient with a mediastinal tumor].

An eleven-year-old girl underwent an open biopsy of a mediastinal tumor extending from the foreneck to the tracheal carina under general anesthesia. Immediately following the bucking developed at the end of the surgery, she fell into serious ventilatory difficulty. A bronchoscopic examination revealed that the tracheal membrane had protruded into the tracheal lumen by its contraction concurring with the bucking, resulting in the obstruction of the endotracheal tube outlet. Endotracheal anesthesia might accompany tracheal stenosis by bucking, particularly in patients with a tumor surrounding the trachea.

Gpr1p, a putative G-protein coupled receptor, regulates glucose-dependent cellular cAMP level in yeast Saccharomyces cerevisiae.

How cells monitor the availability of nutrition and transduce signals is a fundamental, unanswered question. We have found that Gpr1p, a recently identified G-protein (Gpa2p) coupled receptor in yeast Saccharomyces cerevisiae, regulate the cellular cAMP level in response to glucose. The glucose-induced higher cAMP level found in the strain with GPA2 in multicopy plasmid decreased by deletion of GPR1 gene. A transient increase of cAMP in response to glucose was not observed in a Deltagpr1 mutant strain and this defect was complemented and restored by introducing GPR1 gene with YCp vector. Gpr1p was also required for the increase of cAMP in response to other fermentable sugars. Both membrane proximal regions o the third cytosolic loop in Gpr1p, which has been shown to be important for coupling to G-proteins, were also required for glucose-induced transient increase of cAMP. Our findings suggest that Gpr1p is part of the nutrition sensing machinery most likely acting as a receptor to monitor glucose as well as other fermentable sugars and regulate cellular cAMP levels.