Effects of sirtuin 1 activation on nicotine and lipopolysaccharide-induced cytotoxicity and inflammatory cytokine production in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Although sirtuin 1 (SIRT1) over-expression and resveratrol exert anti-inflammatory and proinflammatory effects, their effects and the mechanism of action on human gingival fibroblast (HGF)-mediated inflammation are unknown. The aim of this study was to demonstrate the effects of activating SIRT1 using resveratrol and recombinant adenovirus encoding SIRT1 (Ad-SIRT1) on the expression of proinflammatory cytokines and to elucidate its mechanism of action of lipopolysaccharide (LPS) and nicotine stimulated-HGF. MATERIAL AND METHODS: Cytotoxicity and the production of reactive oxygen species (ROS) were measured using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. The amount of prostaglandin E2 (PGE2 ) released into the culture medium was measured by radioimmunoassay. mRNA and protein levels were analyzed using RT-PCR and western blotting, respectively. RESULTS: Nicotine and LPS up-regulated the expression of SIRT1 mRNA and SIRT1 protein in a time- and concentration-dependent manner. Resveratrol and Ad-SIRT1 decreased LPS and nicotine-induced cytotoxicity, ROS and PGE2 production, and expression of cyclooxygenase-2 in HGFs. Resveratrol and Ad-SIRT1 inhibited nicotine and LPS-mediated protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), p38, ERK, JNK, MAPK and nuclear factor-kappa B (NF-κB) activation. CONCLUSION: This study is the first to show that the anti-inflammatory and cytoprotective effects of SIRT1 activation in HGFs occur through the PKC, PI3K, MAPK and NF-κB pathways.

Lower urinary cotinine level is associated with a trend toward more myopic refractive errors in Korean adolescents.

PurposeTo investigate the association between urinary cotinine levels as an objective biological marker for exposure to nicotine and refractive status.Patients and methodsThis cross-sectional study analyzed data from the Korea National Health and Nutrition Examination Survey between 2008 and 2011. A total of 1139 Korean adolescents aged 12-18 years were enrolled. Urinary cotinine concentrations and other potential risk factors for myopia were examined. Correlation analyses and multivariate regression analysis were performed to investigate the association between urinary cotinine level and refractive error.ResultsSpherical equivalent correlated significantly with urinary cotinine concentration (r=0.104, P=0.011). Lower urinary cotinine level was associated with a trend toward more myopic refractive errors (P for trend=0.003). After adjusting for age, sex, area of residence, physical activity, serum 25-hydroxyvitamin D level, parental income level, and receipt of basic livelihood security, subjects with a low urinary cotinine level had a significantly increased risk of myopia <-0.5 D (odds ratio (OR) 1.95, 95% confidence interval (CI) 1.18-3.21), <-3.0 D (OR 2.03, 95% CI 1.29-3.2), and <-6.0 D (OR 2.2, 95% CI, 1.15-4.23) when compared with subjects with a high urinary cotinine level. As urinary cotinine level decreased, the risks of myopia <-0.5 D, <-3.0 D, and <-6.0 D increased significantly (P for trend <0.05).ConclusionA trend toward less myopic refractive error was observed among Korean adolescents with higher urinary cotinine levels. This result provides the epidemiologic evidence implying nicotine as a potential modulator related with refractive development. Further studies with full consideration for myopia-associated risk factors are required to yield clear answers on the direct effect of smoking to the refractive status.

Prognostic significance of sustained monomorphic ventricular tachycardia induced by programmed ventricular stimulation using up to triple extrastimuli in survivors of acute myocardial infarction.

The prognostic significance of sustained monomorphic ventricular tachycardia (VT) induced by programmed ventricular stimulation using up to 3 extrastimuli was evaluated in 133 consecutive survivors of acute myocardial infarction (AMI) at a mean interval of 1.8 +/- 1.1 months after onset. This was compared with hemodynamic and angiographic abnormalities shown by cardiac catheterization and ventricular ectopic activity detected by Holter monitoring. Sustained monomorphic VT was induced in 25 (19%) patients, sustained polymorphic VT in 11 (8%) patients, nonsustained monomorphic VT (greater than or equal to 10 beats) in 12 patients (9%) and nonsustained polymorphic VT in 9 patients (7%). Multivariate logistic regression analysis of clinical, angiographic, hemodynamic and electrocardiographic variables showed that the presence of a left ventricular aneurysm (p = 0.005) and Lown grade 4B ventricular ectopic activity (p less than 0.001) were independent predictors of inducibility of sustained monomorphic VT. During a mean follow-up of 21 +/- 13 months, there were 8 (6%) sudden cardiac deaths and 3 (2.3%) spontaneous occurrences of life-threatening sustained VT. The 2-year probability of freedom from sudden cardiac death or sustained ventricular tachyarrhythmias was 53 +/- 13% for patients with inducible sustained monomorphic VT, 70 +/- 10% for those with a left ventricular ejection fraction less than 40% and 58 +/- 13% for those with Lown grade 4B ventricular ectopic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Usefulness of early versus late programmed ventricular stimulation in acute myocardial infarction.

To determine the influence of timing on the prognostic value of programmed ventricular stimulation after acute myocardial infarction (AMI), 32 patients were studied on day 19 (early study) and again on day 36 (late study) after AMI using up to 3 extrastimuli. At the early study, sustained monomorphic ventricular tachycardia (VT) was induced in 12 patients (38%), sustained polymorphic VT in 8 (25%), nonsustained monomorphic VT in 1 (3%), nonsustained polymorphic VT in 1 (3%) and no inducible arrhythmia in 10 (31%). At the late study, sustained monomorphic VT, nonsustained monomorphic VT and nonsustained polymorphic VT were induced in 8 patients (25%) each, and no inducible arrhythmia in 8 (25%). Of the 12 patients who had inducible sustained monomorphic VT at the early study, 7 had noninducibility of sustained monomorphic VT at the late study. Of the 20 patients who had noninducibility of sustained monomorphic VT at the early study, 3 had inducible sustained monomorphic VT at the late study. During the follow-up period (mean +/- standard deviation 21 +/- 8 months), there were 2 sudden cardiac deaths and 3 occurrences of sustained VT. Univariate analysis revealed both inducibilities of sustained monomorphic VT at the early study (p = 0.045) and at the late study (p less than 0.001) to be predictive of sudden cardiac death or clinical occurrence of sustained VT. However, inducibility of sustained monomorphic VT at the late study had a higher sensitivity (100%), specificity (89%), positive predictive value (63%) and negative predictive value (100%) than at the early study (80, 70, 33 and 95%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of novel human fetal brain cDNAs mapped to human chromosome bands, 1q25 and 8q24.1.

We isolated chromosome band-specific human fetal brain cDNAs by the microdissection mediated cDNA capture method, and localized these cDNA using in situ hybridization histochemistry with developing rat brain sections. Uni-Amp cDNAs were prepared from an 18-week old human fetal brain, and hybridized to human metaphase chromosomes. Eight Uni-Amp cDNAs, hybridized to chromosome band 1q25 or 8q24.1, were recovered by microdissection and PCR amplification with Uni-Amp primers. Among these cDNAs, two novel genes (FB113 of 8q24.1 and FB134 of 1q25) showed a temporospatially interesting expression pattern in the developing rat brains. The expression of FB113 was under dynamic regulation in the developing granule cells of cerebellum and dentate gyrus. FB134 showed a nervous tissue specific expression pattern and an exclusively prominent expression in the developing presubiculum and parasubiculum. By the fluorescence in situ hybridization using human genomic DNAs, FB113 and FB134 were mapped back to the human chromosome bands 8q24.1 and 1q25, respectively. These results indicate that combined application of the microdissection mediated cDNA capture method and in situ hybridization histochemistry can be used for the isolation of chromosomal band-specific genes related to brain development or human genetic diseases.

Chromosomal aberrations in neuroblastoma cell lines identified by cross species color banding and chromosome painting.

We have studied cytogenetic rearrangements in karyotypes of five neuroblastoma cell lines [SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, SMS-KCNR] by G-banding, cross species color banding (RxFISH), and fluorescence in situ hybridization (FISH) with chromosome painting probes. Each neuroblastoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. The number of rearranged chromosomes in SK-N-AS, SK-N-SH, SH-SY5Y, SK-N-MC, and SMS-KCNR was 11, 3, 7, 14 (tetraploid, 20-21), and 6, respectively. The origins of abnormal chromosomes were effectively analyzed by RxFISH and FISH with multiple chromosome painting probes. The chromosomal origin of the homogeneously staining region in SH-SY5Y was identified as coamplification of chromosome bands 2p13 and 2p24 by chromosome microdissection and FISH. The non-random rearrangements of chromosomes were determined on 1p34 approximately p36, 6q16 approximately q21, 8q24, 9q34, 11q13 approximately q23, 16q23 approximately q24, 17q21, and 22q31. These results may provide useful information for further molecular characterization of neuroblastoma.

Chromosomal aberrations in ovarian malignant brenner tumor cell line using chromosome painting.

Cytogenetic changes in an ovarian malignant Brenner tumor cell line, SNU-840, were investigated by chromosome painting and G-banding. All chromosome alterations were confirmed by the use of multiple chromosome paintings, which also demonstrated a number of additional alterations.

Characterization of chromosomal aberrations in lung cancer cell lines by cross-species color banding.

Using cross-species color banding (RxFISH) and chromosome painting techniques, chromosomal aberrations were investigated in six lung cancer cell lines (NCI-H524, H865, H522, H1373, H358, A549). Each cell line had a variable number of numerical and structural cytogenetic aberrations. While NCI-H524, -H865, and -H522 had near diploidy, NCI-H358, -H1373, and A549 had near triploidy. The origins of the marker chromosomes were further identified by RxFISH and chromosome painting: Nonrandom chromosomal rearrangements were seen on 1p, 3q, 5p10-p15, 6q13-q21, 7q22-q31, 9p32, 15q22-qter, 17p, 17q21-q25, and 21. These abnormal cytogenetic findings indicate that multiple genetic lesions are associated with the development of lung cancer, and thus, these might be possible candidate regions for the abnormal genes involved in lung cancer.

Characterization of chromosomal aberrations in human gastric carcinoma cell lines using chromosome painting.

Using chromosome painting, a study of chromosomal abnormalities was performed in six gastric carcinoma cell lines (SNU-484, 601, 620, 638, 668, 719) from Korean patients. Each carcinoma cell line had unique modal karyotypic characteristics and showed a variable number of numerical and structural clonal cytogenetic aberrations. SNU-484, SNU-620, and SNU-668 had near-triploidy; SNU-601, SNU-638, and SNU-719 had near-diploidy. The origins of the marker chromosomes of these cell lines were identified by fluorescence in situ hybridization with constructed painting probes. In all of six cell lines, rearrangement of chromosome 17 resulting in partial deletion of 17p (and/or partial duplication of 17q) was found. The most frequent marker was a partial gain of chromosome 7 with the breakpoints on 7q22 and 7q31. The nonrandom rearrangements of chromosomes were also determined on 1q32, 5q11-q22, 8q, 14q22, 14q34, and 15q15; suggesting that they may be the candidate regions for the isolation of the genes related to gastric cancer.

Pituitary atrophy in Korean (epidemic) hemorrhagic fever: CT correlation with pituitary function and visual field.

Eleven patients with Korean (epidemic) hemorrhagic fever were each studied three times with high-resolution CT in order to demonstrate necrosis of the pituitary gland and to correlate the CT findings with the patients' pituitary function and visual fields. Seven of the 11 patients showed varying degrees of progressive decrease in the height of the pituitary gland: one severe, two moderate, and four mild. The visual fields of all the patients were checked at the time of the third (last) follow-up CT. Six of the 11 patients had bitemporal superior quadrantanopsia. In five patients, the decreased height (atrophic change) of the pituitary gland and the visual-field defect were coincidental. The visual-field defects in those patients were not improved on follow-up examination 5 weeks later. Two patients in whom a 1-year follow-up examination was performed showed no interval changes in the defects. Pituitary function tests were performed in nine of the 11 patients (six with atrophic pituitary glands and three without atrophic changes) at the time of the third CT. Five of the six patients with atrophy showed decreased pituitary reserve function for follicle-stimulating hormone, cortisol, or human growth hormone, while only one patient showed decreased reserve function for cortisol among the three patients without atrophic change. The pituitary atrophic changes observed on follow-up sellar CT are thought to be the result of the ischemic necrosis of the gland. The high probability (five of seven) of visual-field defects in those patients with atrophic glands suggests optochiasmatic and pituitary ischemia as the basic pathogenesis.

Electrophysiologic characteristics at initiation of ventricular tachycardia and ventricular fibrillation in a canine infarct model.

Local ventricular activation time and the conduction time during sinus rhythm at the induction of ventricular tachycardia (VT) and ventricular fibrillation (VF) were investigated using a canine model of chronic myocardial infarction. Of 26 dogs studied, 15 had inducible VT, 10 had inducible VF, and 1 had no inducible arrhythmias. Bipolar local ventricular electrograms were recorded during sinus rhythm from 136 sites in 10 dogs with VT and 164 sites in 11 dogs with VF. Mean activation time in dogs with inducible VT was significantly longer than in dogs with inducible VF. Furthermore, simultaneous local ventricular electrograms were recorded during the induction of VT (74 episodes) or VF (38 episodes) from the infarct border zone at the endocardium (B-EN), the epicardium (B-EP), and normal sites (N-EN, N-EP). During VT induction, the activation time at N-EN and N-EP was significantly longer than during VF induction (N-EN: 94 +/- 21, 70 +/- 19 ms; N-EP: 83 +/- 21, 64 +/- 10 ms; p < 0.05). Conduction time was measured at the initiation of VT or VF induced by orthodromic or antidromic pacing. The conduction times of the last paced beat between N-EN and B-EP (35 +/- 11, 62 +/- 24 ms), N-EN and N-EP (35 +/- 12, 14 +/- 13 ms), B-EN and B-EP (16 +/- 10, 38 +/- 25 ms), and B-EP and N-EP (77 +/- 27, 44 +/- 12 ms) were significantly different in dogs with inducible VT (p < 0.05), but not in dogs with VF.(ABSTRACT TRUNCATED AT 250 WORDS)

A large myxoma of the right atrium demonstrated by thallium-201.

A rare case of right atrial myxoma in which thallium-201 gave a good delineation of the tumor was presented. In this case, the feeding arteries were seen to be highly developed on coronary arteriogram. The amount of blood containing thallium-201 supplied to the tumor through the feeding arteries was so great that the tumor was considered to be visualized by thallium-201 imaging.

[Effect of verapamil on myocardial infarct size estimated by serial 201-thallium single-photon emission tomography].

To investigate the effects of intravenous verapamil (V) in coronary thrombolytic therapy, we serially observed the time course of perfusion of the myocardium by 201-thallium (Tl) SPECT in patients who were successfully reperfused within 6 hours from the onset. 201-Tl SPECT was attempted serially on the 1st-2nd day, the 7th-10th day and 28th-30th day. In addition to this, we calculated the count ratio of radioactivity of 99mTc-PYP (CR) in the infarcted myocardium to sternum to evaluate intracellular uptake of calcium during reperfusion. The infarct size, estimated by % Defect decreased significantly in the patients treated with V, while it remained unchanged in the patients without it. In the patients with V, the left ventricular ejection fraction was more favourable, and exercise-induced ischemia determined by redistribution of 201-Tl SPECT in the chronic phase was found more frequently. CR showed no difference between reperfused myocardium irrespective of the treatment. In conclusion, verapamil was considered to enhance myocardial salvage carried out by reperfusion, and not to affect the influx of intracellular calcium into the injured myocytes.

Estimating mineral changes in enamel formation by ashing/BSE and microCT.

Enamel formation produces the most highly mineralized tissue in the human body. The growth of enamel crystallites is assisted by enamel proteins and proteinases. As enamel formation progresses from secretory to maturation stages, the composition of the matrix with its mineral and non-mineral components dynamically changes in an inverse fashion. We hypothesized that appropriately calibrated micro-computed tomography (µCT) technology is suitable to estimate the mineral content (weight and/or density) and volume comparable in accuracy with that for directly weighed and sectioned enamel. Different sets of mouse mandibular incisors of C57BL/6 mice were used for dissections and µCT reconstructions. Calibration phantoms corresponding to the range of enamel mineral densities were used. Secretory-stage enamel contained little mineral and was consequently too poor in contrast for enamel volumes to be accurately estimated by µCT. Maturation-stage enamel, however, showed remarkable correspondence for total mineral content per volume where comparisons were possible between and among the different analytical techniques used. The main advantages of the µCT approach are that it is non-destructive, time-efficient, and can monitor changes in mineral content of the most mature enamel, which is too physically hard to dissect away from the tooth.

Cleavage site specificity of MMP-20 for secretory-stage ameloblastin.

Ameloblastin is processed by protease(s) during enamel formation. We tested the hypothesis that MMP-20 (enamelysin) catalyzes the cleavages that generate secretory-stage ameloblastin cleavage products. We isolated a 23-kDa ameloblastin cleavage product from developing enamel and determined its N-terminus sequence. Ameloblastin was stably expressed and secreted from HEK293-H cells, purified, and digested with MMP-20 or Klk4 (kallikrein 4). The digests were analyzed by SDS-PAGE and Western blotting, and cleavage products were characterized by N-terminal sequencing. Six fluorescent peptides were digested with MMP-20 and Klk4 and analyzed by RP-HPLC and by mass spectrometry. MMP-20 cleaved each peptide exactly at the sites corresponding to ameloblastin cleavages catalyzed in vivo. Klk4 cleaved ameloblastin and the fluorescent peptides at sites not observed in vivo, and cleaved at only a single correct site: before Leu(171). We conclude that MMP-20 is the enzyme that processes ameloblastin during the secretory stage of amelogenesis, and we present a hypothesis about the sequence of ameloblastin cleavages.

Transgenic rescue of enamel phenotype in Ambn null mice.

Ameloblastin null mice fail to make an enamel layer, but the defects could be due to an absence of functional ameloblastin or to the secretion of a potentially toxic mutant ameloblastin. We hypothesized that the enamel phenotype could be rescued by the transgenic expression of normal ameloblastin in Ambn mutant mice. We established and analyzed 5 transgenic lines that expressed ameloblastin from the amelogenin (AmelX) promoter and identified transgenic lines that express virtually no transgene, slightly less than normal (Tg+), somewhat higher than normal (Tg++), and much higher than normal (Tg+++) levels of ameloblastin. All lines expressing detectable levels of ameloblastin at least partially recovered the enamel phenotype. When ameloblastin expression was only somewhat higher than normal, the enamel covering the molars and incisors was of normal thickness, had clearly defined rod and interrod enamel, and held up well in function. We conclude that ameloblastin is essential for dental enamel formation.

In vitro differential developmental toxicity of vitamin A congeners.

Several forms of vitamin A were tested in the in vitro hydra assay for their developmental toxicity hazard potential and site of action on progressive ontogenesis. Retinol, retinyl acetate, retinaldehyde, all trans retinoic acid, and 13 cis retinoic acid were tested fully, and each was established clearly as being able to perturb development of artificial hydra "embryos" at, or near, adult toxic treatment levels. All forms of vitamin A tested interfered with differentiation, but, although the alcohol, acetate, and aldehyde forms (group I) prevented the initial stages of differentiation from occurring, the acid forms (group II) allowed the initial stages of differentiation to occur but not the final differentiation of tentacle buds. Group I compounds produced the developmental toxicity endpoint after as little as 24 h of transient exposure on the first day of development, but had no permanent effect on development at their minimal affective developmental concentration (D-MAC) when exposure began after the first day of development. In contrast, transient 24-h exposure to group II forms did not interfere with development. At, or even above, a concentration greater than the D-MAC, more continuous exposure to them was required to interfere with differentiation. Consistent with tests of other chemicals, the concentrations needed to produce effects in hydra bore no relation to those needed to produce effects in mammals.

Relationship of developmental stage to effects of vinblastine on the artificial "embryo" of Hydra.

Three milligrams of vinblastine sulfate per liter of culture medium prevented normal development of all reaggregated artificial hydra "embryos" at the 66-hour stage of regeneration. One milligram of vinblastine per liter of medium had no effect on development, and 100 mg did not markedly accelerate the developmental disruption beyond the pace of the 2 mg/liter concentration which was the lowest effective concentration. Treatment with vinblastine through the first few hours of development was sufficient to produce the toxic endpoint at 66 hours of incubation, but effects were delayed in appearance when treatment was initiated later in development. Early development of exposed preparations proceeded normally even in the presence of rather high concentrations of the test substance, and the pellet was resistant to the effects of vinblastine treatment begun after 24 hours of development. From these results, it would appear that interference with cell division is not the primary means by which vinblastine perturbs this developing system.

Regeneration by dissociated adult Hydra cells: a histologic study.

Adult Hydra attenuata with vitally stained gastrodermal cells were dissociated into their component cells which were then randomly reaggregated into pellets by low-speed centrifugation. Representative examples of these preparations, which develop into normal adult hydra if left undisturbed, were examined fresh at low magnification and at higher magnification in fixed, stained, and sectioned specimens. The actual pellet stage lasts less than 1 hour because the adult ectodermal and gastrodermal cells rapidly sort themselves into an inner and outer layer and seem to secrete a new mesoglea immediately thereafter. The "embryo" becomes trilaminar and attains a central cavity by extruding a large amount of cellular debris at the end of the first day. At about this same time, new tentacles begin to differentiate from rapidly dividing and undifferentiated interstitial cells. Regulation of tentacle number and position occurs at the end of two days, and the body form is essentially reestablished within 60 hours by further differentiation of the hypostomes and body wall. Complete separation of the preparation into individual polyps does not occur until about 190 hours of development.