Inhibition of NADPH oxidase 4 activates apoptosis via the AKT/apoptosis signal-regulating kinase 1 pathway in pancreatic cancer PANC-1 cells.

Pancreatic adenocarcinoma is an aggressive human malignancy and is characterized by resistance to apoptosis. Recently, NADPH oxidase (Nox) 4-mediated generation of intracellular reactive oxygen species (ROS) was proposed to confer antiapoptotic activity and thus a growth advantage to pancreatic cancer cells. The signaling mechanism by which Nox4 transmits cell survival signals remains unclear. Here, we show that both a flavoprotein inhibitor, diphenylene iodonium (DPI), and small interfering RNAs designed to target Nox4 mRNA (siNox4RNAs) inhibited superoxide production in PANC-1 pancreatic cancer cells, and depletion of ROS by DPI or siNox4RNAs induced apoptosis. Parallely, DPI treatment and siNox4RNA transfection blocked activation of the cell survival kinase AKT by attenuating phosphorylation of AKT. Furthermore, AKT phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) on Ser-83 was reduced by DPI and siNox4RNAs. When ASK1Ser83Ala (an AKT phosphorylation-defective ASK1 mutant) was introduced into PANC-1 cells, this mutant alone induced apoptosis. But, addition of DPI or co-transfection of siNox4RNA had no additive effect, indicating that the mutant can substitute for these reagents in apoptosis induction. Taken together, these findings suggest that ROS generated by Nox4, at least in part, transmit cell survival signals through the AKT-ASK1 pathway in pancreatic cancer cells and their depletion leads to apoptosis.

Expression of coxsackievirus and adenovirus receptor in hearts of rats with experimental autoimmune myocarditis.

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Lack of association between lumbar disc degeneration and osteophyte formation in elderly japanese women with back pain.

Our study was designed to assess the contributions of the physical and constitutional factors to osteophyte formation, disc degeneration, and bone mineral density (BMD) in lumbar vertebrae of elderly postmenopausal women. A total of 126 Japanese women with back pain, aged over 60 years, were invited to participate in the study. Then 80 subjects with a full set of data for physical examinations, radiographs, MRI, and DXA were examined. TaqI polymorphism of vitamin D receptor (VDR) gene was examined in 60 subjects. Prevalence rates of osteophytes (on radiographs) and disc degeneration (on MRI) were 61 and 68%, respectively. Body weight and BMI correlated significantly with anteroposterior (AP) and lateral (LAT) BMD (r = 0.354 for weight, r = 0.347 for BMI) and mean osteophyte area (r = 0.557 for weight, r = 0.486 for BMI), and body weight also correlated with number of discs with osteophytes. However, these did not correlate with the disc area or the number of degenerated discs. Stepwise regression analysis revealed that body weight and LAT-BMD values independently related to the osteophyte area. Disc area (r = 0.386 for AP view) and osteophyte area (r = 0.384 for AP view) significantly correlated with BMD. However, disc area and osteophyte area did not correlate with each other (r = 0.056). The proportion of degenerated discs was higher in the lower lumbar discs, but not the proportion of discs with osteophytes. Frequencies of T and t alleles of VDR did not correlate with disc degeneration, osteophyte formation, or osteoporosis. Our data showed that increases in osteophyte formation and BMD in the lumbar vertebrae are influenced by body weight and BMI, but did not correlate with disc area, which correlated inversely with BMD. Disc degeneration and osteophyte formation seem to represent two different factors that affect lumbar spine in elderly women.

Methotrexate maintains bone mass by preventing both a decrease in bone formation and an increase in bone resorption in adjuvant-induced arthritic rats.

We examined the effects of low doses methotrexate (MTX) and indomethacin (IND) on bone mass and turnover in normal male Sprague-Dawley rats and those with adjuvant-induced arthritis. Normal and the adjuvant (heat-killed mycobacterium)-injected rats, 6 weeks of age, were given MTX at daily doses of 0.05, 0.1, or 0.2 mg/kg body weight (BW) or IND at a daily dose of 1.0 mg/kg BW. Rats were killed at the start, or at 14 and 28 days. In normal rats, the administration of these agents did not change the lumbar and femoral BMD values, nor did the serum osteocalcin or urinary deoxypyridinoline (D-Pyr) levels. Lumbar trabecular osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS) were decreased in the rats given IND. In the arthritic rats, the administration of MTX did not prevent an early increase of paw edema in the adjuvant-injected limb, but late inflammatory edema was alleviated in the non-injected limb. However, MTX administration at a dose of 0.1-0.2 mg/kg BW maintained an age-dependent increase in the lumbar and femoral BMD values. While serum osteocalcin levels were decreased and urinary D-Pyr values were increased in the arthritic control rats, these bone markers remained at the levels of the normal rats. Decreases in mineral apposition rate (MAR) and bone formation rate (BFR/BS) and increases in the trabecular Oc.N/BS and Oc.S/BS values were prevented by MTX. While IND almost completely prevented inflammatory paw edema, it did not improve the parameters of bone formation. An increase in osteoclasts was prevented and the osteopenia in the lumbar and the femoral bone was only partially prevented by IND. These data suggest that MTX improves bone mass and turnover in the arthritic rat, in which several cytokines that affect bone cells are involved. An increase in bone resorption may be due to prostaglandins, but bone formation defect was suggested to be due to other cytokines such as IL-1, IL-6, and TNF-alpha in this model.

Effect of atropine on QT prolongation and torsade de pointes induced by intracoronary acetylcholine in the long QT syndrome.

We recently reported a marked QT prolongation and torsade de pointes (TDP) induced by an intracoronary acetylcholine (ACh) administration in patients with long QT syndrome, but the mechanism was not determined. In the present study, the effect of atropine on the ACh-induced QT prolongation and TDP was studied in long QT syndrome. Nine patients with congenital long QT syndrome were studied. ACh at doses of 20, 50, and 100 microg were injected in a stepwise manner into the left main coronary artery, and the changes in the QT interval were measured. In 4 of the 9 patients, ACh administration at a dose of 100 microg was repeated after an intravenous atropine administration at a dose of 0.5 mg. The QT intervals were measured using 12-lead electrocardiograms, and the data were compared before and after atropine administration. The coronary angiograms were normal and coronary spasm was not induced by ACh in all patients. The intracoronary administration of ACh at a dose of 100 microg significantly prolonged the corrected QT interval (QTc), from 511 +/- 26 to 629 +/- 40 ms (p <0.05). In 5 of the 9 patients, TDP was induced and was spontaneously terminated within 10 seconds (n = 4) or required direct-current shock (n = 1). After atropine administration, intracoronary ACh at the same dose resulted in no QT prolongation, and the QTc interval remained unchanged (525 +/- 29 vs 520 +/- 21 ms before and after atropine), and no TDP was induced. These findings indicate that the muscarinic receptor is involved in ACh-induced QT prolongation and TDP, both of which were prevented by the atropine administration.

Recovery of the right atrial effective refractory period after cardioversion of chronic atrial fibrillation.

The effective refractory period was shorter in patients with than without chronic atrial fibrillation (AF). The effective refractory period was prolonged, and at 12 and 24 hours after cardioversion of AF it was the same as the subjects without AF.

Triterpene alcohol and sterol ferulates from rice bran and their anti-inflammatory effects.

Six novel feruloyl esters of triterpene alcohols and sterols, viz., two trans-ferulates, cycloeucalenol and 24-methylenecholesterol trans-ferulates, and four cis-ferulates, cycloartenol, 24-methyelenecycloartanol, 24-methylcholesterol, and sitosterol cis-ferulates, besides five known trans-ferulates, cycloartenol (CAR), 24-methylenecycloartanol (24-MCA), 24-methylcholesterol, sitosterol, and stigmastanol trans-ferulates, and one known cis-ferulate, stigmastanol cis-ferulate, were isolated from the methanol extract of edible rice bran. These and eight other synthetic trans- and cis-ferulates of triterpene alcohols and sterols, along with the corresponding free alcohols, were evaluated with respect to their anti-inflammatory activity against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation (1 microg per ear) in mice. All of the ferulates showed marked inhibitory activity, and their 50% inhibitory dose (ID(50)) was 0. 1-0.8 mg per ear. On the other hand, whereas two free triterpene alcohols, CAR and 24-MCA, showed strong inhibition (ID(50) 0.2-0.3 mg/ear), eight free sterols examined showed weaker activity (ID(50) 0.7-2.7 mg/ear) than their corresponding ferulates.

[Assessment of the amount of drug deposited in the lungs of asthmatic children using disodium cromoglycate as the marker].

Disodium cromoglycate (DSCG) is easily absorbed from the airways and lungs, and is excreted unchanged in the urine and bile. Therefore it is possible to estimate the dose of DSCG deposited in the airways and lungs based upon urinary excretion. The urinary concentration of DSCG was measured by the HPLC method in 78 asthmatic children aged 0 to 16 years after they had inhaled 20 mg nebulizer solutions with facemasks. Jet-type nebulizers were used. The mean urinary excretion of DSCG in the patients aged 0, 1, 2 and 3 years from 4-hour urinary collection represented 0.204%, 0.231%, 0.593%, 0.790%, respectively, of the dose administered. In the patients aged 3, 4, 5-6, 7-9 and 10-16 years, from 24-hour urinary collection, the figures were 0.625%, 0.895, 0.855, 1.176%, 1.070%, respectively. The mean dose deposited in the airways and lungs of the age groups 0-1, 2, 3, 4-6, 7-9, and 10-16 years represented approximately 0.5%, 1.4%, 1.4-1.8%, 2.0%, 2.7%, 2.4%, respectively, of the dose administered. Although there was a wide range in the total amount of DSCG deposited in the airways and lungs of asthmatic children, these data seem to provide a useful guide to standardizing the dosing in inhalation therapy.

[Diagnostic value of glass microfibre-based basophil histamine release test in food allergic children. Comparison with specific IgE antibody and skin scratch test].

We evaluated the diagnostic value of the glass microfibre-based histamine release test (HRT), which allows measurements to be performed using small amounts of whole blood, in 50 children with food allergy case histories. The patients were evaluated by radioallergosorbent tests (RAST), skin scratch tests (ST) and food challenge tests. Of the 50 patients, 39 had a confirmed clinical diagnosis of food allergy from food challenge tests and case histories, and were affected by a total of 60 positive allergens (egg 37, milk 11, soy beans 4, wheat 5, rice 3). The concordance, sensitivity, and specificity of HRT with the clinical diagnosis were 85.3%, 66.7% and 92.1%, those of RAST were 59.4%, 90.0% and 48.2%, and those of ST were 84.7%, 71.8% and 88.7%, respectively. The positive predictive values of HRT, RAST and ST were 75.5%, 38.8% and 66.7%. The false positive ratio of HRT (24.5%) was the lowest among all the tests. There was a significant correlation between HRT and RAST (r = 0.513, p < 0.001). However, the concordance of HRT with respect to RAST was 56.0%. The concordance and specificity of HRT in relation to the clinical diagnosis were higher than RAST and the same as ST. The sensitivity of RAST was higher than that of HRT. From these results, we concluded that RAST is good for the screening of allergens and that HRT is a useful diagnostic method for the confirmation of a clinical allergy.

Quantification and detection of bacteria from postoperative maxillary cyst by polymerase chain reaction.

BACKGROUND/AIMS: Postoperative maxillary cyst (POMC) is known to occur as a delayed complication of radical maxillary sinus surgery, such as Caldwell-Luc surgery. The cyst gradually expands with no symptoms over a period of years, and then occasionally causes swelling and pain in the buccal region and/or the mucogingival fold. It is probable that bacterial infection affects the progression of POMC symptoms. The aims of this study were to determine the bacterial density and to examine the presence of 20 oral bacteria in POMC fluids. METHODS: POMC fluids (4 purulent, 2 mucous and 4 serous) were sampled from 10 subjects (aged 43-77 years). Bacterial quantification and detection were performed by real-time polymerase chain reaction (PCR) and nested PCR based on bacterial 16S rRNA genes, respectively. RESULTS: Bacterial DNA was detected in all samples and the average concentrations of bacterial DNA were 5.9 (purulent), 0.5 (mucous), and 0.7 (serous) ng/mg of sample. Twelve bacterial species, including anginosus streptococci, known to be associated with abscess formation, were detected in the purulent fluids, while two and five species were detected in the mucous and serous fluids, respectively. CONCLUSION: Purulent fluids contained numerous bacteria of various types, thus suggesting that oral bacteria may cause symptoms such as pain in POMC with purulent fluids. Mucous and serous fluids also contained bacteria, although their numbers were small, thus suggesting an association between bacteria and progression of POMC.

Reentrant ventricular tachycardia. Interpretations of electrophysiologic findings and its applications.

Monomorphic sustained ventricular tachycardia (MSVT) was revisited in relation to the electrophysiologic findings and their relation to the drug efficacy. Old myocardial infarction is less common cause of MSVT in Japan, and the majority (about 2/3) of MSVT is unrelated to coronary artery disease but, the mechanism shared a common mechanism: reentry with an excitable gap as others. The reentrant mechanism was supported from the inducibility, the terminability of VT by electrical stimulation, and by the ability to entrain with rapid pacing. In MSVT associated with underlying heart diseases, diseased myocardium showed low amplitude and fragmented electrograms and the area was considered to participate as the central common pathway of reentrant circuit. The area of slow pathway showed a decremental conduction or all-or-nothing conductive property. The width of the excitable gap seemed to be determined by the maximal conductive frequency but not by the duration of action potential: effective refractory period. As to the drug efficacy, there was no baseline characteristics in predicting the efficacy. However, the significant narrowing of the width of the excitable gap was associated with the drug efficacy and VT became non-inducible after addition of the same drug. The response pattern of the excitable gap to specific drug including class III, was not predictable. Further electropharmacological studies will be warranted.

Molecular cloning and nucleotide sequence of the cDNA for human liver glutamate dehydrogenase precursor.

Two cDNA clones (lambda GDHh1 and lambda GDHn61) for glutamate dehydrogenase (GDH) were isolated from a human liver cDNA library in lambda gt11. The clone, lambda GDHh1, was isolated from the library using a synthetic 45mer oligodeoxy-ribonucleotide, the sequence of which was derived from the known amino acid sequence near the NH2-terminus of human liver GDH. Subsequently, lambda GDHn61 was isolated from the same library using lambda GDHh1 as a probe. The inserts of both clones contained an overlapping cDNA sequence for human liver GDH, consisting of a 5'-untranslated region of 70 bp, an open reading frame of 1677 bp, a 3'-untranslated region of 1262 bp and a 15 base poly(A) tract. The predicted amino acid sequence revealed that the human liver GDH precursor consisted of a total of 558 amino acid residues including the NH2-terminal presequence of 53 amino acids. The sequence deduced for the mature enzyme showed 94% homology to the previously reported amino acid sequence of human liver GDH.

The displacement of the femoral head by rotational acetabular osteotomy. A radiographic study of 97 subluxated hips.

To determine the limits of medial and inferior displacement of the subluxated femoral head by rotational acetabular osteotomy, we studied the acetabular coverage and position of the femoral head radiographically before and after surgery in 97 hips. The median age of the patients at the time of surgery was 33 (18-54) years. The position of the femoral head was represented by its center and medial and upper borders. The average increase and decrease in the CE and the AC angle were 39 degrees and 27 degrees, respectively. The average medial displacement of the head was 8 (-12 to +19) mm measured from its center, and 7 (-10 to +21) mm measured from its medial border. The average inferior displacement was 5 (-6 to +19) mm from its center and 4 (-10 to +15) mm from the upper border. These results indicate that concentric reduction by rotational acetabular osteotomy is limited and that medial displacement of the subluxated femoral head is within similar ranges obtained by other conventional pelvic osteotomies.

New Aldobiuronic Acid, 3-O-α-D-Glucopyranuronosyl-L-rhamnopyranose, from an Acidic Polysaccharide of Chlorella vulgaris.

The new aldobiuronic acid, 3-O-α-D-glucopyranuronosyl-L-rhamnopyranose, was isolated from the acid hydrolyzate of an acidic polysaccharide in cells of Chlorella vulgaris K-22. Its structure was elucidated by NMR spectroscopic analyses.

A comparison of electrophysiologic properties between responders and non-responders to DL-sotalol among patients with ventricular tachyarrhythmia. Importance of the lack of a reverse use-dependent effect on ventricular refractoriness to responders.

This study was undertaken to determine whether dl-sotalol can prevent ventricular tachyarrhythmia inducibility that can be predicted from electrophysiologic parameters. The effects of dl-sotalol in 16 patients (ventricular tachycardia (VT) in 11 and fibrillation (VF) in 5) were determined in electrophysiologic studies before and after dl-sotalol (320 mg/day). In 9 of 16 patients (56%) after dl-sotalol, ventricular tachyarrhythmia could not be induced by the entire stimulation protocol (responders). There were significant differences in QT interval (462 +/- 52 vs. 415 +/- 34 msec; p < 0.05) and ventricular effective refractory period (VERP) at 600, 400 and 300 msec (302 +/- 28 vs. 262 +/- 20 msec; p < 0.001, 280 +/- 23 vs. 240 +/- 21 msec; p < 0.001, 256 +/- 24 vs. 222 +/- 12 msec; p < 0.005, respectively) between responders and non-responders. The percentile increases in VERP (% VERP) at 600, 400, and 300 msec in responders were 25%, 26%, and 27%, whereas those in non-responders was 9%, 7%, and 7%, respectively. Isoproterenol administered to responders did not fully reverse the dl-sotalol-induced prolongation of VERP (delta VERP) at 600, 400, and 300 msec, which remained significantly prolonged compared to the baseline (281 +/- 18 vs. 241 +/- 16 msec; p < 0.01, 258 +/- 20 vs. 223 +/- 21 msec; p < 0.01, 247 +/- 22 vs. 202 +/- 16 msec; p < 0.01, respectively). % VERP did not exhibit significant differences at 600 (16%), 400 (15%), and 300 (20%) msec, indicating the lack of a reverse use-dependency. The results suggest that delta VERP in responders did not show reverse use-dependency, and that the phenomenon may account for the efficacy of dl-sotalol.

Nested PCR for detection of mutans streptococci in dental plaque.

AIMS: Mutans streptococci such as Streptococcus mutans and Streptococcus sobrinus have been implicated in human dental caries. In an attempt to develop a rapid and sensitive method for detecting Strep. mutans and Strep. sobrinus in dental plaque, a nested PCR amplification based on the 16S rRNA gene was employed. METHODS AND RESULTS: A universal set of PCR primers for bacterial 16S rRNA gene was introduced for the first PCR, and then two sets of primers specific for the 16S rRNA gene sequences of either Strep. mutans or Strep. sobrinus were used for the second PCR. Eighteen plaque samples were analyzed, and a nested PCR was shown to be more sensitive for detecting Strep. mutans and Strep. sobrinus than direct PCR. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The 16S rRNA gene-based nested PCR method is a rapid and sensitive method for the detection of mutans streptococci, and may also be suitable for carrying out large-scale studies on the cariogenicity of mutans streptococci.

Abrupt loss of constant fusion during entrainment of ventricular tachycardia at a critical paced cycle length.

Sustained monomorphic ventricular tachycardia (VT) can be frequently entrained and interrupted with rapid pacing and the mechanism of the pacing-induced interruption is considered to be due to orthodromic block. This study focused on the incidence of VT which was interrupted at a critical cycle length and was characterized by an abrupt loss of constant fusion in the surface electrocardiogram (ECG), and the role of orthodromic block as the cause of such characteristic change and interruption of VT was analyzed. Among 45 consecutive patients with symptomatic VT, rapid pacing was performed in 43 VTs of 39 patients. The exit was mapped as the earliest site of the activation during VT and an electrode catheter was located at the site. Rapid pacing was performed at progressively shorter cycle lengths in steps of 10 msec until VT was interrupted and the timing of the orthodromic and direct capture was compared at the exit. Abrupt loss of constant fusion was observed in 25 of 39 patients (64.1%): and the loss was invariably associated with interruption of VT. When the timings of the activation of the exit were compared, which were measured from the preceding (n-1) stimulus as the time reference, the direct capture was relatively delayed compared to that of the orthodromic capture. This finding suggests that orthodromic block is the cause of the direct capture as well as the pacing-induced interruption of VT. In the remaining 13 patients (35.9%), the surface ECG showed a gradual transition into the fully paced QRS morphology. The direct capture was confirmed in the non-fused beats, but it was not necessarily associated with interruption of VT. The interval from the stimulus to the entrained electrogram at the exit showed a gradual prolongation until the exit was finally captured directly from the pacing site. The confirmation of constant fusion followed by abrupt loss in ECG can be a reliable hallmark of orthodromic block as the cause of the interruption of VT during transient entrainment at a critical paced cycle length.

The effect of 17 beta-estradiol treatment on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin.

We performed the dosing experiment to establish whether estrogen administration has any beneficial effects on the mass and the turnover of bone in ovariectomized rats taking a mild dose of thyroxin. Thirty-five Wistar rats, 28 weeks of age, received ovariectomies (OVX) or sham operations and were divided into five groups. Group 1 was the sham group, Groups 2-5 were ovariectomized. Group 2 was the OVX-control, Group 3 treated with thyroxin 30 micrograms/kg/day (T4), Group 4, 17 beta-estradiol 0.3 mg/kg/week (E2), and Group 5, the combination of T4 and E2. The duration of the experiment was 12 weeks. At the end of the experiment, serum chemistries were measured. Bone minerals in the femur were determined with single photon absorptiometry and bone turnover was assessed histomorphometrically. Alkaline-phosphatase increased in Group 3 (OVX-T4), but it reduced in Groups 4 (OVX-E2) and 5 (OVX-T4 + E2). Bone minerals decreased in Groups 2 (OVX) and 3. In Group 4, it was preserved at the same level as in Group 1. Group 5 showed a significant increase of bone mass compared with Group 1. Eroded surface and osteoid surface increased in Groups 2 and 3 and they were reduced in Groups 4 and 5. Bone volume and mineral apposition rate were at a maximum in Group 5. This study demonstrated that 17 beta-estradiol was capable of preventing the bone mass decrease by regulating the turnover in ovariectomized rats taking a mild dose of thyroxin. Osteoblast function appeared to be stimulated in combination with 17 beta-estradiol and thyroxin.