Isolation and characterization of dental epithelial cells derived from amelogenesis imperfecta rat.

OBJECTIVE: Disruption of the third zinc finger domain of specificity protein 6 (SP6) presents an enamel-specific defect in a rat model of amelogenesis imperfecta (AMI rats). To understand the molecular basis of amelogenesis imperfecta caused by the Sp6 mutation, we established and characterized AMI-derived rat dental epithelial (ARE) cells. MATERIALS AND METHODS: ARE cell clones were isolated from the mandibular incisors of AMI rats, and amelogenesis-related gene expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Localization of wild-type SP6 (SP6WT) and mutant-type SP6 (SP6AMI) was analyzed by immunocytochemistry. SP6 transcriptional activity was monitored by rho-associated protein kinase 1 (Rock1) promoter activity with its specific binding to the promoter region in dental (G5 and ARE) and non-dental (COS-7) epithelial cells. RESULTS: Isolated ARE cells were varied in morphology and gene expression. Both SP6WT and SP6AMI were mainly detected in nuclei. The promoter analysis revealed that SP6WT and SP6AMI enhanced Rock1 promoter activity in G5 cells but that enhancement by SP6AMI was weaker, whereas no enhancement was observed in the ARE and COS-7 cells, even though SP6WT and SP6AMI bound to the promoter in all instances. CONCLUSION: ARE cell clones can provide a useful in vitro model to study the mechanism of SP6-mediated amelogenesis imperfecta.

Association between cardiac function and pulmonary function in hypertensive patients.

OBJECTIVE: This study examined the association between cardiac function and pulmonary function in hypertensive patients. METHODS: Hypertensive patients without overt cardiovascular disease were enrolled (n=43; mean±SD age 71±9 years). Pulmonary function was measured by the percentage of predicted forced vital capacity (%FVC) and the ratio of 1 s forced expiratory volume (FEV1) to FVC (FEV1/FVC ratio). Left ventricular ejection fraction (LVEF) and the ratio of peak early diastolic transmitral flow (E) to peak early diastolic mitral annular velocity (e') (E/e' ratio) were assessed using echocardiography. RESULTS: Multiple linear regression analysis revealed that E/e' was independently associated with %FVC and that LVEF was independently associated with FEV1/FVC ratio. Both LVEF and FEV1/FVC ratio were significantly lower in hypertensive former or current smokers than in hypertensive never smokers. CONCLUSIONS: Subclinical cardiac dysfunction was independently associated with reduced pulmonary function in hypertensive patients. Hypertensive patients with decreased pulmonary function may need preventive care to prevent the progression of heart failure.

X-ray waveguide mode in resonance with a periodic structure.

We present a novel concept for x-ray waveguiding based on electromagnetism in photonic crystals, using a waveguide consisting of a pair of claddings sandwiching a core with a periodic structure. By confining the x rays undergoing multiple interference in the core by total reflection, a characteristic waveguide mode whose field distribution matches the periodicity of the core is formed. The distinctively low propagation loss enables the single-mode propagation of x rays. This concept opens broad application possibilities in x-ray physics from coherent imaging to x-ray quantum optics.

Differences in left ventricular diastolic dysfunction between eccentric and concentric left ventricular hypertrophy in hypertensive patients with preserved systolic function.

Left ventricular (LV) hypertrophy (LVH) may be eccentric or concentric (2 × LV posterior wall thickness relative to LV end-diastolic dimension ≤ 0.42 or > 0.42, respectively). The LV diastolic function between age-matched hypertensive patients with eccentric and concentric LVH was compared in the present study. Echocardiography was used to measure LV mass index (LV mass/body surface area; LVMI) as an index of LVH. LV diastolic function was assessed by measurements of peak early transmitral flow velocity (E)/peak late transmitral flow velocity (A) (the E/A ratio), peak early diastolic mitral annular velocity (e') and the E/e' ratio. Although LVMI, E/A and e' did not differ between the two groups, E/e' was significantly higher (worse) in patients with concentric LVH (13.4 ± 5.4) than in those with eccentric LVH (11.1 ± 3.6). Among hypertensive patients with LVH, those with concentric LVH may, therefore, have more severe LV diastolic dysfunction than those with eccentric LVH even if their LVMIs, which reflect the degree of LVH, are similar.

Aortic root dilatation as a marker of subclinical left ventricular diastolic dysfunction in patients with cardiovascular risk factors.

Consensus is lacking about the clinical importance of aortic root dilatation in assessment of the risk of cardiovascular disease. In this study, correlations between aortic root diameter and echocardiographic features of left ventricular (LV) diastolic function were investigated in 333 patients with at least one cardiovascular risk factor (hypertension, diabetes or dyslipidaemia) and preserved LV systolic function. Aortic root diameter was measured by M-mode echocardiography, and LV diastolic function was evaluated by measuring the peak velocity of early (E) and late (A) diastolic transmitral blood flow and peak early diastolic mitral annular velocity (E') by Doppler echocardiography. Linear regression analysis showed that, in men, age was not related to aortic root diameter but hypertension and LV hypertrophy were, whereas the converse was true in women. The parameters E, E/A ratio and E', were related to aortic root diameter in both sexes. Stepwise multiple regression analysis confirmed that E in women and E' in men were independently associated with aortic root diameter. It is concluded that aortic root dilatation might be a useful marker of subclinical LV diastolic dysfunction. Patients with preserved systolic function showing aortic root dilatation should, therefore, be given preventative therapy against LV diastolic heart failure.

Induction of peripheral mononuclear cell apoptosis in asthmatic patients in remission.

Apoptosis regulates inflammatory cell survival in allergic inflammation, and decreased apoptosis contributes to the chronicity of inflammation. To investigate the mechanisms of onset and remission of mite-sensitive childhood asthma, we evaluated peripheral blood mononuclear cell apoptosis in patients with asthma and in remission. There was a similar percentage of hypodiploid cells in unstimulated mononuclear cell cultures from patients with active asthma (29.5+/-5.0%) and normal individuals (25.9+/-4.9%). In contrast, the percentage increased in patients in remission (44.5+/-3.2%). In Dermatophagoides farinae (Df) antigen-stimulated mononuclear cell, the stimulation index was lower in patients with active asthma (0.95+/-0.06%) than in normal individuals (1.31+/-0.16%). In contrast to active patients, the proportion of hypodiploid cells stimulated with Df in patients with remission was equivalent to that of normal controls. After phytohemaglutinin (PHA) stimulation, the percentage of hypodiploid cells in patients with active asthma (35.1+/-3.2%) was also lower than in normal individuals (48.5+/-4.3%) or patients in remission (49.5+/-5.7%). Apoptosis occurred predominantly in CD8+, but not CD4+, cells in patients in remission. Interleukin IL-2 inhibited apoptosis in Df-activated cells in normal individuals, whereas IL-2 did not inhibit apoptosis in cells from patients in remission as well as with active asthma. The expression of Fas receptors on resting mononuclear cells was similar in the three groups. However, Fas receptor expression in Df-stimulated mononuclear cells was greater in patients with active asthma than in healthy individuals. In patients with remission that was equivalent to healthy controls. The PHA increased Fas expression to a similar degree in the three groups. With regard to Fas ligand, the expression was lower in unstimulated cultured mononuclear cells from patients than in normal individuals. In patients in remission that was comparable to normal individuals. The Df stimulation upregulated the Fas ligand in patients with active asthma, and downregulated it in patients in remission. In conclusion, apoptosis in Df-stimulated mononuclear cells is impaired in patients with active asthma, while spontaneous apoptosis of CD8+ cells in vivo is augmented in patients in remission, and may be involved in the onset and remission of mite-sensitive asthma.

Functional human IgE specific for Dermatophagoides farinae antigen is produced in SCID mice reconstituted with peripheral mononuclear cells derived from healthy persons and patients with asthma.

BACKGROUND: Whether normal peripheral blood mononuclear cells (PBMCs) transferred to severe combined immunodeficient (SCID) mice produce specific IgE remains unclear. METHODS: Mice received injections of Dermatophagoides farinae antigen (Df)-stimulated PBMCs from healthy persons (IgE RAST score of 0). RESULTS: High titers of Df-specific IgE were detected. The Df-specific IgE activity produced was comparable to or higher than that produced by cells from patients with asthma although the time to maximal production was longer. IgE derived from PMBCs of healthy persons or patients with asthma induced histamine release from cultured human basophils that had been stimulated with Df antigen or an anti-IgE antibody. Treatment of Df-stimulated PBMCs with a high dose, but not a low dose, of interleukin-4 stimulated production of Df-specific IgE by PMBCs from healthy persons or patients with asthma. In contrast, intravenous injection of IFN-gamma into reconstituted SCID mice decreased Df-specific IgE production by PBMCs from patients with asthma. In PMBCs from healthy persons, IgE class-switching may occur later and block the effects of treatment with IFN-gamma. CONCLUSIONS: PBMCs from healthy persons and persons with asthma have clones reactive to allergen and produce functional IgE specific for relevant antigens in mite-sensitive bronchial asthma.

Inverse correlation between macrophage-colony stimulating factor, cholesterol and high density lipoprotein cholesterol in Kawasaki disease.

Kawasaki disease (KD) is a childhood-onset vascular disease. We assessed the concentrations of macrophage-colony stimulating factor (M-CSF) and those of lipids in sera from patients with KD. The M-CSF concentration in patients with acute-phase KD was 2,914+/-159 U/ml, significantly higher than that in control subjects with Infectious diseases (1,241+/-96 U/ml). The elevated levels of this cytokine in the acute phase fell to 1,319+/-138 U/ml in the convalescent phase. Total and high-density lipoprotein cholesterol concentrations in acute phase KD (113.8+/-8.4 and 21.5+/-2.3 mg/dl, respectively) were lower than in the infectious disease controls (195.8+/-7.0 and 62.5+/-1.8 mg/dl). The elevation of M-CSF correlated with the decrease of total and high-density lipoprotein cholesterol. Overproduction of macrophage-colony stimulating factor activates macrophages and monocytes and may disturb the lipid metabolism. Both effects could contribute to vasculitis in KD.

Pioglitazone improves left ventricular diastolic function and decreases collagen accumulation in prediabetic stage of a type II diabetic rat.

This study investigated the effect of pioglitazone, an insulin sensitizer, on metabolic abnormalities and oxidative stress as a cause of myocardial collagen accumulation in prediabetic rat hearts. Twenty male diabetic rats and 9 male nondiabetic age-matched rats were used. The diabetic rats were divided into two groups: diabetic treated and untreated. Pioglitazone was mixed in rat chow fed to the diabetic treated group (0.01%). Treatment duration was 5 weeks. At baseline (15 weeks) and 20 weeks of age, blood glucose, lipid, insulin, and plasma malondialdehyde-thiobarbituric acid (MDA) levels were measured and Doppler echocardiography was tracked. At 20 weeks of age, left ventricular collagen content was studied. Blood glucose, plasma insulin, and triglyceride levels in the diabetic treated group were significantly lower than those in the untreated diabetic group. Deceleration time (ms) of early diastolic inflow in the treated diabetic group decreased significantly compared with the untreated diabetic group (65 +/- 8 vs. 77 +/- 8, p < 0.01). Ratio of left ventricular weight to body weight (mg/g) and ratio of left ventricular collagen content to dry weight (mg/100 mg) were decreased in the treated diabetic group (1.5 +/- 0.1, 1.3 +/- 0.3) compared with the untreated diabetic group (1.7 +/- 0.2, p < 0.01; 1.7 +/- 0.3, p < 0.05). Plasma MDA concentration (nmol/ml) significantly decreased (2.9 +/- 0.3 at baseline to 2.3 +/- 0.3 at 20 weeks, p = 0.001) in the treated diabetic group, and was lower than that in the untreated diabetic group (3.2 +/- 0.7 at 20 weeks, p < 0.05). Pioglitazone improved glucose and lipid metabolism and reduced oxidative stress in the left ventricle, which decreased left ventricular collagen accumulation and improved left ventricular diastolic function of prediabetic rat hearts.

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix.

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6 kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42 degrees C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.

Inhibition by eicosapentaenoic acid of oxidized-LDL- and lysophosphatidylcholine-induced human coronary artery smooth muscle cell production of endothelin.

The objectives of the present study were (1) to determine whether oxidized low-density lipoprotein (LDL) and lysophosphatidylcholine (lyso-PC), a major phospholipid component of oxidized LDL, stimulate the production of endothelin-1 (ET)-1 in cultured human coronary artery smooth muscle cells (SMCs), and (2) to examine the possible effect of an antiatherogenic agent, eicosapentaenoic acid (EPA), on oxidized-LDL- and lyso-PC-stimulated ET-1 production in these cells. Oxidized LDL (10-50 microg/ml) and lyso-PC (10(-7) to 10(-5) mol/l) stimulated ET-1 production in a concentration-dependent manner. By contrast, the effects of native LDL and phosphatidylcholine were modest or absent. Lyso-PC (10(-7) to 10(-5) mol/l) and oxidized LDL (10-50 microg/ml) significantly induced particulate protein kinase C (PKC) activation. Lyso-PC- and oxidized-LDL-stimulated ET-1 production was significantly inhibited by PKC inhibitor, PKC (19-36). EPA (80-160 micromol/l) clearly suppressed ET-1 production stimulated by oxidized LDL and lyso-PC in a concentration-dependent manner. Furthermore, EPA (160 micromol/l) significantly inhibited lyso-PC (10(-5) mol/l)- and oxidized LDL (50 microg/ml)-induced particulate PKC activation. Results suggest that oxidized LDL and lyso-PC stimulate ET-1 production by a mechanism involving activation of PKC, and that EPA suppresses ET-1 production stimulated by lyso-PC as well as oxidized LDL probably through the modulation of PKC in human coronary artery SMCs. EPA may exert an antiatherosclerotic effect, in part, through these mechanisms.

Enhanced expression of matrix metalloproteinase-9 in abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAAs) are characterized by structural alterations of the aortic wall resulting from degradation of collagen and elastin. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, show strong elastinolytic activity. We examined the levels of mRNA for MMP-2, MMP-9, membrane type (MT)-MMP-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 in AAAs (n = 8), atherosclerotic occlusive diseases (AOD) (n = 8), and normal subjects (n = 8) using the reverse transcription-polymerase chain reaction (RT-PCR). We also analyzed the gelatinolytic activity of these metalloproteinases using gelatin zymography. The levels of MMP-2 and MMP-9 mRNA were increased in the AAA group compared with those in the AOD group and normal subjects. The levels for TIMP-1 and TIMP-2 mRNA in the AAA group were also higher than those in the AOD and normal groups. Only in the case of MT-MMP-1 was the difference between AAA and AOD not statistically significant. By gelatin zymography with the same samples used for RT-PCR, gelatinolytic activity of MMP-9 was elevated in all AAA tissues. The 62-kDa form of MMP-2 was elevated in both the AAA and AOD groups and did not differ significantly between them. Linear regression analysis demonstrated a significant positive correlation between mRNA levels of MMPs and those of TIMPs. These observations suggest that aneurysm formation in patients with atherosclerosis is related to the degree of MMP-9 expression.

Effect of roxithromycin on T lymphocyte proliferation and cytokine production elicited by mite antigen.

Clinical evidence suggests that roxithromycin (RXM) may be an effective additional therapy for bronchial asthma. However, how it interferes with allergic responses is unclear. To investigate the mechanisms of action of RXM, lymphocyte transformation and interferon (IFN)-gamma, interleukin (IL)-4 and IL-5 synthesis associated with Dermatophagoides farinae (Df), mite antigen in patients with bronchial asthma were evaluated in vitro in the presence of RXM. T cell proliferation in Df antigen-stimulated patients' lymphocytes was suppressed by 50-100 microg/ml of RXM. Production of IL-4 and IL-5 was similarly decreased by 1-10 microg/ml RXM, whereas, IFN-gamma production, which was reduced by Df-stimulation alone, was increased by 50 microg/ml RXM. Our results suggest that skewed cytokine profiles of patients with mite antigen-induced bronchial asthma may be corrected with RXM, which may mimic those of patients in remission, who are tolerant of Df antigen.

Quantitative analysis of telomerase activity and telomerase reverse transcriptase expression in renal cell carcinoma.

To investigate the relationship between the telomerase activity levels and clinicopathological features of tumors, we quantified the telomerase activities of 23 renal cell carcinomas (RCCs) and four non-cancerous tissues, using a modified telomeric repeat amplification protocol assay, and assessed the hTERT mRNA levels of these samples by reverse transcription-polymerase chain reaction analysis. Elevated levels of telomerase activity had correlation with tumor stages as well as the degree of nuclear grades. Our findings suggested that telomerase activity is a useful indicator for tumor aggressiveness in RCCs. However, hTERT mRNA levels in RCCs had no correlation with nuclear grades and tumor stages. The telomerase activities and the hTERT mRNA levels in cancer cells were not always in parallel. These results suggested that telomerase activity is regulated in a posttranscriptional manner as well as a post-translational manner in tumor cells.

Successful immunogene therapy using colon cancer cells (colon 26) transfected with plasmid vector containing mature interleukin-18 cDNA and the Igkappa leader sequence.

IL-18 is a novel cytokine that induces interferon (IFN)-gamma secretion and plays an important role in antitumor immunity. In the present study, we constructed plasmid vectors encoding the murine mature IL-18 cDNA linked with the Igkappa leader sequence and the pro-IL-18 cDNA to estimate the efficacy of the mature IL- 18 vector and to evaluate IL-18--producing tumor cells as a tumor vaccine. Colon 26 cells were transfected with the abovementioned vectors or with vector alone (mock). Reverse transcription-polymerase chain reaction analysis showed increased expression of murine IL-18 cDNA in both mature IL-18 and pro-IL-18 transfectants in comparison to that in mock transfected cells. The ability of the culture supernatants of mature IL-18 transfectants to induce IFN-gamma secretion was extremely high (40-140 pg/10(6) cells) in comparison to that of pro-IL-18 transfectants (4-18 pg/10(6) cells). When injected into syngeneic BALB/c mice, the growth of mature IL-18 transfectants, but not pro-IL-18 transfectants, was significantly less than that in mock transfected cells ( P< .01, by ANOVA and analysis of covariance). In addition, injection of colon 26 or Meth-A cells into mice immunized with a mature IL-18 transfectant revealed acquired immunity. Depletion of natural killer cells did not affect the growth of transfectants. However, the growth inhibitory effects were partially abrogated following treatment with anti-CD4+ and anti-CD8+ antibodies. These data suggest that the rejection of mature IL-18/colon 26 cells was mediated through T-cell activation. Gene therapy using mature IL-18 transfectants containing a plasmid vector and the Igkappa leader sequence may be a useful tumor vaccine.

Improvement of left ventricular diastolic dynamics in prediabetic stage of a type II diabetic rat model after troglitazone treatment.

Troglitazone, an oral antidiabetic agent, has hypoglycemic effects in insulin-resistant animal models and humans. This study was conducted to evaluate its effect on left ventricular diastolic dynamics of a spontaneous diabetic (DM) rat model. Twenty DM rats and 20 age-matched nonDM rats were used, and 10 of each group were treated with troglitazone as a 0.2% food admixture for 10 weeks. At 5 and 15 weeks of age, Doppler echocardiography and M-mode echocardiography were performed. Troglitazone treatment significantly improved the left ventricular diastolic dynamics of DM rats: deceleration time (msec) of early diastolic inflow decreased significantly (treated 52 +/- 3 vs untreated 64 +/- 5, p = 0.0002), and peak velocity of early transmitral inflow (cm/sec) increased significantly (treated 96 +/- 7 vs untreated 86 +/- 8, p = 0.0216). The data suggest that troglitazone improves left ventricular diastolic dynamics of a DM rat model at prediabetic stage.

Fatal buckwheat dependent exercised-induced anaphylaxis.

Cases of food-dependent exercise-induced anaphylaxis (FEA) caused by buckwheat have been rare. Clinical, laboratory, and autopsy findings are present on an 8-year old girl with FEA caused by Japanese buckwheat. The patient consumed buckwheat noodles called "zaru soba" and immediately thereafter swam vigorously. Approximately 30 minutes later, she complained of abdominal pain, vomiting, coughing, and chest discomfort. Another ten minutes later her consciousness level deteriorated and she experienced cardiorespiratory arrest. The heart beat was restored and she was admitted to the hospital. She never regained consciousness and expired after another arrest 13 days later. Her IgE level was high (2,840 IU/ml) and the IgE-radioallergosorbent test (RAST) score was 2 for soybeans, 3 for buckwheat, 2 for rice, and 3 for wheat. An exaggerated hematemesis that occurred immediately after hospital admission indicated an inflammatory condition of the digestive tract that was caused by buckwheat. Marked ulceration accompanied with hemorrhage and necrosis was noted at the ileum. Extensive hemorrhage involving the endotracheal pulmonary field and lymphocyte infiltration of the alveolar space likely appeared after the inflammation. The analysis of buckwheat-specific IgE antibody by immunoblotting showed 7 bands that reacted with the IgE of the patient's serum, 4 bands: 16, 20, 24, and 58 kDa, were specific to the patient as compared to subjects not allergic to buckwheat. A first case of fatal FEA by buckwheat is reported with reference to specific IgE.