Bone morphogenetic protein signaling mediated by ALK-2 and DLX2 regulates apoptosis in glioma-initiating cells.

Bone morphogenetic protein (BMP) signaling exerts antitumor activities in glioblastoma; however, its precise mechanisms remain to be elucidated. Here, we demonstrated that the BMP type I receptor ALK-2 (encoded by the ACVR1 gene) has crucial roles in apoptosis induction of patient-derived glioma-initiating cells (GICs), TGS-01 and TGS-04. We also characterized a BMP target gene, Distal-less homeobox 2 (DLX2), and found that DLX2 promoted apoptosis and neural differentiation of GICs. The tumor-suppressive effects of ALK-2 and DLX2 were further confirmed in a mouse orthotopic transplantation model. Interestingly, valproic acid (VPA), an anti-epileptic compound, induced BMP2, BMP4, ACVR1 and DLX2 mRNA expression with a concomitant increase in phosphorylation of Smad1/5. Consistently, we showed that treatment with VPA induced apoptosis of GICs, whereas silencing of ALK-2 or DLX2 expression partially suppressed it. Our study thus reveals BMP-mediated inhibitory mechanisms for glioblastoma, which explains, at least in part, the therapeutic effects of VPA.

Cloning and characterization of the cDNA encoding human biliverdin-IX alpha reductase.

Biliverdin reductase is classified into two isoforms in substrate specificity; biliverdin-IX alpha reductase and biliverdin-IX beta reductase with a molecular mass of 22 kDa and 34-42 kDa, respectively. We have cloned the cDNA encoding human biliverdin-IX alpha reductase from MOLT4 cDNA library. The cDNA of 1146 bp in nucleotide length contained an entire reading frame coding 296 amino acid residues. The NADH/NADPH binding consensus sequence was found in the amino-terminal region. Comparison between human and rat biliverdin-IX alpha reductases showed 82.8% identity in amino acid sequences and 80.3% identity in the coding nucleotides. The amino acid sequence of human biliverdin-IX alpha reductase showed no significant homology to that of human biliverdin-IX beta reductase. Northern blot analysis of poly(A) RNA from eight different human tissues revealed that the reductase mRNA was abundant in the brain, lung and pancreas but not in the liver. The distribution pattern of biliverdin-IX alpha message was different from that of heme oxygenase activity which is known to be high in the liver and to be low in the heart and lung.

Three percent diquafosol ophthalmic solution as an additional therapy to existing artificial tears with steroids for dry-eye patients with Sjögren's syndrome.

PURPOSE: To investigate the long-term results of 3% diquafosol ophthalmic solution as an alternative therapy to existing ophthalmic solutions, including topical immunosuppression, for the treatment of dry eye in patients with Sjögren's syndrome. METHODS: This study involved 14 female dry-eye patients (mean age: 62.4 years) with Sjögren's syndrome who insufficiently responded to their current therapy. In all patients, 3% diquafosol ophthalmic solution was administered six times daily for 12 months in substitution for artificial tears and sodium hyaluronate ophthalmic solution. Their use of corticosteroid eye drops remained unchanged from that prior to the treatment with diquafosol sodium. The subjective symptoms assessed, and ocular signs including tear meniscus radius and the tear film breakup time, and ocular-surface epithelial damage score were examined at 1, 2, 3, 4, 5, 6, 9, and 12 months after initiating treatment. RESULTS: Among the subjective symptoms, significant improvement was obtained in dryness at 2 months post treatment, in eye fatigue at 1, 2, 3, 4, and 12 months post treatment, and in pain at 1, 2, 6, and 12 months post treatment. Difficulty in opening the eye, foreign body sensation, and redness were also significantly ameliorated at various time-points. The tear meniscus radius and the tear film breakup time were significantly improved throughout the observation period, and the corneal epithelial staining scores were significantly decreased at 3 months post treatment. CONCLUSIONS: In dry-eye patients with Sjögren's syndrome, treatment with 3% diquafosol ophthalmic solution improved both symptoms and signs, and that effectiveness was maintained for 12 months.

Cell death during corneal storage at 4 degrees C.

PURPOSE: To evaluate cell death in human donor corneas stored at 4 degrees C, to determine whether terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4 degrees C. METHODS: Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4 degrees C for periods ranging from 0 to 21 days and then fixed for histologic examination. Central corneal sections from each cornea were examined by transmission electron microscopy (TEM) and by the TUNEL assay. Electron micrographs of at least 15 keratocytes each from the anterior, middle, and posterior stroma were examined by three masked observers who graded each cell as normal, apoptotic, or necrotic. Central sections from the same corneas were processed by the TUNEL assay and evaluated with a laser scanning confocal microscope to determine the percentage of apoptotic cells. RESULTS: By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes, with the results in individual corneas being similar to the findings by TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis occurred in 13% of the epithelial cells and in 8% of the endothelial cells. The percentage of apoptotic cells and storage time correlated significantly for the epithelium, but not for the keratocytes or endothelium in this small sample. CONCLUSIONS: Both apoptosis and necrosis occur in cells during corneal storage at 4 degrees C, with apoptosis appearing to predominate. The TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneal tissue. Inhibition of apoptosis in corneas stored at 4 degrees C may prolong acceptable storage times.

Influence of food thickness and hardness on possible feed-forward control of the masseteric muscle activity in the anesthetized rabbit.

The facilitatory masseteric muscle response (FMR) elicited by polyurethane foam strip application between the opposing molars during cortically-induced rhythmic jaw movements (CRJMs) was induced earlier than masticatory force onset. The occurrence of this early response of the FMR (e-FMR) could not be explained by a simple reflex mechanism. One possible mechanism of the e-FMR is the involvement of a feed-forward control mechanism of the masticatory jaw movement. In the present study, experimentally designed polyurethane foam strips with various thickness and hardness were applied during CRJMs and analyzed in terms of how the e-FMR was modulated by the food hardness and thickness. The FMR onset was not related to the strip thickness or the strip hardness. However, the magnitude of the e-FMR increased in a thickness and a hardness-dependent manner. The sensory information of the food properties in the masticatory cycle may make the FMR adequate to chewing of the food in the following cycle, and such modulation may help chewing rhythms remain stable.

Characteristics of the muscle spindle endings of the masticatory muscles in the rabbit under halothane anesthesia.

To explore the response characteristics of muscle spindle units in the masticatory muscles in the rabbit, the responses of muscle spindle units were recorded from the mesencephalic trigeminal nucleus (MesV) under halothane anesthesia during ramp-and-hold stretches. Three firing patterns, initial burst (IB) at the onset of the dynamic phase, negative adaptation (NA) at the end of the dynamic phase and firing during the release (FDR) phase, were observed during muscle stretch. IB was present at higher stretch velocities, FDR at lower stretch velocities. The velocity at which an IB or FDR was present was different from unit to unit. Because, within the range of the velocities of stretch tested, units with NA always showed NA and units without NA never did, all recorded units were divided into two groups on the basis of the existence of NA (NA(+) or NA(-) units). Response characteristics of the two groups were then compared. NA(+) units showed an IB more frequently and FDR less frequently than NA(-) units. NA(+) units had significantly higher dynamic responsiveness and discharge variability than NA(-) units. The conduction velocity of the afferents of NA(+) units was higher than that of NA(-) units. However, distributions of these measurements were not bimodal. These results suggest that NA is the useful criteria to classify the muscle spindle endings in the masticatory muscles in the rabbit under halothane anesthesia.

Prolonged impairment of peripheral corneal epithelium barrier function after successful trabeculectomy.

PURPOSE: To quantitate changes in the ocular surface after trabeculectomy using a newly designed fluorophotometer-based method. METHODS: We studied 23 normal eyes (23 volunteers), six eyes (six patients) within 1 month after surgery (early group), and 14 eyes (14 patients) 3 months or longer after surgery (late group). Also, to elucidate time-dependent changes in corneal epithelium barrier function, we measured fluorescein uptake before and after trabeculectomy prospectively in the central and peripheral cornea of six glaucomatous eyes in six patients. RESULTS: Mean fluorescein uptake (+/- SE) in the central cornea 30 minutes after instillation was 34.4 +/- 3.8 ng/ml, 113.5 +/- 65.8 ng/ml, and 43.1 +/- 12.8 ng/ml, respectively, in the normal, early, and late groups, which represents a statistically significant increase in the early group, but subsequent recovery to near normal range in the late group (P = .0225, normal vs early groups; P = .437, normal vs late groups, unpaired t test). Mean fluorescein uptake in the peripheral cornea was 34.5 +/- 6.1 ng/ml, 276.2 +/- 120.5 ng/ml, and 378.4 +/- 118.2 ng/ml, respectively, in the normal, early, and late groups, which represents a statistically significant increase in both the early and late groups (P = .0267, normal vs early groups; P = .0310, normal vs late groups, unpaired t test). In the early and late groups, significant differences were shown in fluorescein uptake between the central and peripheral cornea, but these differences were not evident in the normal group. The prospective study showed that fluorescein uptake in the peripheral cornea was 35.3 +/- 13.0 ng/ml and increased to 308.2 +/- 96.6 ng/ml at the third postoperative month (P = .022, paired t test) and to 277.9 +/- 145.5 ng/ml at the end of the first postoperative year (P = .154, paired t test). CONCLUSION: Peripheral corneal epithelium barrier function adjacent to the filtration bleb following trabeculectomy may be significantly impaired for at least 1 year after surgery.

Effectiveness of hyaluronan on corneal epithelial barrier function in dry eye.

AIMS/BACKGROUND: The aim of this study was to assess quantitatively the effectiveness of hyaluronan on corneal disruption in patients with dry eye. Corneal epithelial barrier function was evaluated by measuring fluorescein permeability using a slit-lamp fluorophotometer. METHODS: 11 patients with dry eye were assigned to this study. Hyaluronan ophthalmic solution (0.1% hyaluronic acid) was instilled five times a day to the right eye, in addition to the usual artificial tear solutions. The left eye received only the artificial tear solutions. Corneal barrier function was evaluated on the pretreatment day, and at 2 and 4 weeks after treatment. Fluorophotometry was used to measure fluorescein uptake at the central and lower corneal portions. RESULTS: Two weeks after treatment, hyaluronan treated right corneas showed significant corneal epithelial barrier improvement in the lower portion, compared with the pretreatment day (p < 0.025). Four weeks after treatment, the treated corneas showed significant improvement in the central corneal portion (p < 0.025) and improvement in the lower portion, compared with the pretreatment day. The untreated left corneas, on the other hand showed no improvement during the course of the study. CONCLUSION: This study suggests that hyaluronan is effective in the treatment of corneal epithelial disruption in dry eye.

Epithelial barrier function of the filtering bleb conjunctiva and the cornea after trabeculectomy with mitomycin C.

PURPOSE: To evaluate epithelial barrier function of the filtering bleb conjunctiva and the cornea after trabeculectomy with adjunct, intraoperative mitomycin C, by using a newly developed slit-lamp fluorophotometer. SUBJECTS AND METHODS: Thirteen patients with normal-tension glaucoma with a cystic filtering bleb who had undergone unilateral trabeculectomy with mitomycin C only once at least 1 year previously were subjected to the study. Epithelial barrier function of the bleb conjunctiva and the cornea in the operated eye was evaluated using an Anterior Fluorometer FL-500 to measure the uptake of topically applied fluorescein. RESULTS: Fluorescein uptake (nanograms per milliliter, mean +/- standard error) by the bleb conjunctiva (1,857 +/- 380) was not significantly different from either the uptake by the superior bulbar conjunctiva in the operated eyes, located approximately 90 degrees C away from the filtering bleb (1,974 +/- 258), or the uptake by the conjunctiva in the intact eyes, located symmetrically against the bleb (1,913 +/- 248). The corneal uptake in the operated eye (56.1 +/- 10.2) was not significantly different from that in the intact eye (53.5 +/- 8.3). CONCLUSIONS: Epithelial barrier function of the cystic filtering bleb conjunctiva and the cornea long after trabeculectomy with mitomycin C was demonstrated to be almost identical to that of the control.

Effects of aldose reductase inhibitor CT-112 on the corneal epithelial barrier of galactose-fed rats.

PURPOSE: To investigate whether the barrier function of the corneal epithelium is disrupted in galactosemic rats, and to assess the effects of the aldose reductase inhibitor CT-112, in the form of eyedrops, on the corneal epithelial barrier in galactosemic rats. METHODS: Forty rats were divided into 3 groups based on their diet: a control group, a galactose group and a CT-112 treated galactose group (CT-112 group). After 3 weeks, 31 rats from the 3 groups were subjected to fluorophotometry, in which fluorescein (F) was instilled into one eye and carboxyfluorescein (CF) was instilled into the other eye in a random fashion. The F and CF uptakes were then measured at the central cornea by a slit-lamp fluorophotometer. Three rats from each group were exposed to a horseradish peroxidase (HRP) solution for one hour, and the HRP-reactive substances within the corneal epithelium were also examined via electron microscopy. RESULTS: There was significantly higher F uptake in the galactose group than in the control (p = 0.003) and CT-112 groups (p = 0.028). There were no significant differences in CF uptake between the 3 groups. Histologically, HRP-reactive substances were found in much greater quantities within the superficial corneal cells of the galactose group than in the control or CT-112 groups. CONCLUSIONS: These results suggest that cell membrane disruption, as detected by F uptake and HRP penetration, was found in the superficial corneal cells of galactose-fed rats, and that intercellular junction integrity can be assayed by CF uptake and histological evaluation. Moreover, CT-112 eyedrops were effective in improving the corneal epithelial barrier dysfunction of galactose-fed rats.

[Corneal epithelial disturbance during topical use of latanoprost].

PURPOSE: To report two cases with corneal epithelial disturbance during topical use of the new prostaglandin F 2 alpha derivative latanoprost and a beta-blocker. CASES: Both case 1 (82-year-old male) and case 2 (50-year-old female) were treated with topical antiglaucoma drugs for ocular hypertension. FINDINGS: One month (case 1) and 10 days (case 2) after treatment with topical beta-blocker and latanoprost, corneal epithelial disturbance occurred. The corneal epithelial disturbance improved over several weeks with the preservative-free artificial tears after discontinuation of latanoprost and the beta-blocker. CONCLUSION: Careful observation is necessary during the course of treatment with latanoprost and a beta-blocker.

[Quantitative evaluation of corneal epithelial barrier function in corneal epithelial edema].

Corneal epithelial barrier function in patients with corneal epithelial edema was evaluated by fluorophotometry. Fluorescein uptake through the intact corneal epithelium was 28.1 +/- 2.7 ng/ml (n = 35, mean +/- SE (standard error), calculated from the standard line) at 30 minutes after instillation of 3 microliters of 0.5% sodium fluorescein (20 minutes after eye washing with 20 ml of BSS PLUS). Fluorescein uptake by the cornea in 7 patients with corneal epithelial edema and 6 patients with corneal stromal edema without epithelial edema were 2,489.7 +/- 1,045.6 ng/ml and 99.3 +/- 25.0 ng/ml, respectively, showing a significant increase when compared to normal corneal epithelium (p < 0.0005). Moreover, in patients with corneal epithelial edema, the corneal epithelial barrier function showed significant decrease in comparison with those having corneal stromal edema without epithelial edema (p < 0.05). These results demonstrate a significant decrease in the corneal epithelial barrier function in the epithelial edema.

[Evaluation of conjunctival epithelial damage in dry eye].

Using sulphorhodamine B, which is highly sensitive in detecting damaged ocular surface epithelium, we evaluated conjunctival epithelial damage in dry eye patients. The subjects were 99 eyes of 50 dry eye patients (41 eyes of 21 patients with Sjögren's syndrome and 58 eyes of 29 keratoconjunctivitis sicca patients without Sjögren's syndrome). We also investigated the relation between sulphorhodamine B and fluorescein staining in ocular surface epithelium. The conjunctival epithelial damage stained with sulphorhodamine B showed a high correlation with corneal epithelial damage stained with fluorescein. In addition, conjunctival damage in Sjögren's syndrome tended to be more severe than in patients without Sjögren's syndrome.

[Comparison of primary and secondary Sjögren's syndrome].

PURPOSE: We studied the difference in severity between primary and secondary Sjögren's syndrome (SS). SUBJECTS AND METHODS: Two groups of patients (all females, mean age: 58 years), 31 with primary SS and 18 with secondary SS were studied. We performed the following dry eye tests: fluorescein score and Rose Bengal staining, grading of tear lipid layer interference patterns, measurement of fluorescein break up time, cotton thread test, and Schirmer-I test. Auto antibodies were also investigated. RESULTS: There was no significant difference between primary and secondary SS with respect to any dry eye tests or auto antibodies. In primary SS, however, the presence of anti SS-A antibody was significantly correlated with Rose Bengal scores (p = 0.044). CONCLUSION: The severity of SS is independent of the primary or secondary type. In primary SS, the presence of anti SS-A antibody may be correlated with the severity.

[Effect of latanoprost on the barrier function of corneal epithelium].

PURPOSE: We evaluated the effect on corneal epithelium barrier function of instillation of prostaglandin F2 alpha ophthalmic solution (latanoprost) for one month. MATERIALS AND METHODS: Ten healthy volunteers and nine glaucoma patients were enrolled in this study. The barrier function was determined as uptake of topically applied sodium fluorescein by the central cornea measured with an anterior fluorophotometer(FL-500, Kowa Co. Ltd). Healthy volunteers and glaucoma patients received 0.005% latanoprost instillation once daily for one month. We measured the uptake of fluorescein by the cornea of each subject before and one month after instillation. RESULTS: Fluorescein uptake before the instillation was 22.2 +/- 16.0 ng/ml (mean +/- standard deviation) and 26.4 +/- 15.1 ng/ml one month after the treatment in the normal group, and it was 55.0 +/- 25.0 ng/ml before treatment and 57.8 +/- 37.0 ng/ml after treatment in the glaucoma group. There was no significant difference in the uptake of fluorescein before and after treatment in either of two groups. CONCLUSION: These results suggested that the barrier function of corneal epithelium was not compromised after the instillation of latanoprost for at least one month.

[Pulmonary metastasis of the stomach cancer].

Despite the fact that the lungs have a plenty of blood and lymphatic supply, only 20-40% of pulmonary metastasis from the stomach cancer are found. Pulmonary metastases are most commonly described as well-circumscribed lesions complicated with hilar metastasis. However, the most important picture is that of diffuse lymphatic permeation, which may produce clinical and radiologic features of lymphangitis carcinomatosa showing a characteristic roentgenologic appearance, namely, a fine feathery interstitial streaking. Furthermore, pleuritis carcinomatosa is a common type of metastasis from the stomach cancer and often presents a difficult management problem. Instillation of cytotoxic or sclerosing agents into the pleural space can be effective in preventing recurrence.

TGF-ß-induced apoptosis of B-cell lymphoma Ramos cells through reduction of MS4A1/CD20.

Transforming growth factor-ß (TGF-ß) exhibits growth inhibitory effects on various types of tumor cells, including B-cell lymphoma cells. In the present study, the role of TGF-ß in the survival of Epstein-Barr virus-negative B-cell lymphoma Ramos cells was investigated. As TGF-ß-induced apoptosis of Ramos cells in vitro and in vivo, we attempted to identify novel target gene(s) responsible for their survival. Oligonucleotide microarray analysis and chromatin immunoprecipitation revealed that Smad proteins directly regulated the transcription of membrane-spanning 4-domains, subfamily A, member 1 (MS4A1), also known as CD20, in Ramos cells upon TGF-ß stimulation. In addition, immunohistochemical analysis using clinical samples from B-cell lymphoma patients showed an inverse correlation between the expression of MS4A1/CD20 and phosphorylation of Smad3. Although knockdown of MS4A1/CD20 in Ramos cells resulted in an increase of apoptotic cells, Ramos cells stably expressing MS4A1/CD20 were resistant to TGF-ß-induced apoptosis. This suggests that MS4A1/CD20 is responsible for TGF-ß-induced apoptosis of B-cell lymphoma cells. Moreover, downregulation of MS4A1/CD20 by TGF-ß attenuated the effects of the monoclonal anti-MS4A1/CD20 antibody, rituximab, on Ramos cells. Our findings suggest that the sensitivity of B-cell lymphoma cells to rituximab may be affected by TGF-ß signaling.

Transforming growth factor-ß decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-ß (TGF-ß) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-ß repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-ß, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-ß negatively contributes to maintain the CICs within the cancer.

Radio frequency ion source operated with field effect transistor based radio frequency system.

Characteristics of radio frequency (RF) plasma production are investigated using a field effect transistor inverter power supply as an RF wave source. With the frequency of around 0.3 MHz, an electron density over 10(18) m(-3) is produced in argon plasma. Although lower densities are obtained in hydrogen plasma, it drastically increased up to 5x10(18) m(-3) with an axial magnetic field of around 100 G applied in the driver region. Effects of the magnetic field and gas pressure are investigated in the RF produced plasma with the frequency of several hundred kilohertz.