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.

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.

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.

Propofol pharmacokinetics in a dwarfism patient.

Pharmacokinetic information is important to control anesthetic depth. However, there are few available pharmacokinetic data of propofol in dwarfism patients. We anesthetized a dwarfism patient who underwent spinal decompression, and investigated the pharmacokinetics of propofol. The patient was a 40-year-old man suffering from muscle weakness and numbness in the arms. The operation consisted of two stages; anterior approach in the supine position and posterior approach in the prone position. We also obtained arterial blood for pharmacokinetic analysis. Distribution volume at steady-state and clearance in the supine position was 180 and 0.92 l min- 1, respectively, and in the prone position 127 and 0.74 l min- 1, respectively, in spite of a continuous infusion of dopamine. The data in the supine position were well predicted by Gepts' parameters (used in Diprifusor Zeneca Ltd, Cheshire, UK), which means the target-controlled infusion (TCI) technique can be available in the supine position, while attention is necessary to avoid overdosing when a patient is placed in the prone position.

Putative feed-forward control of jaw-closing muscle activity during rhythmic jaw movements in the anesthetized rabbit.

When a thin plastic test strip of various hardness is placed between the upper and lower teeth during rhythmical jaw movements induced by electrical stimulation of the cortical masticatory area (CMA) in anesthetized rabbits, electromyographic (EMG) activity of the masseter muscle is facilitated in a hardness-dependent manner. This facilitatory masseteric response (FMR) often occurred prior to contact of the teeth to the strip, and thus preceded the onset of the masticatory force. Since this finding suggests involvement of a feed-forward mechanism in the induction of the FMR, the temporal relationship between the onset of the FMR and that of the masticatory force was analyzed in five sequential masticatory cycles after application of the strip. The FMR was found to precede the onset of masticatory force from the second masticatory cycle after application of the strip, but never did in the first cycle. This finding supports the concept of a feed-forward control mechanism that modulates FMR timing. Furthermore, the FMR preceding the force onset disappeared after making a lesion of the mesencephalic trigeminal nucleus (MesV) where the ganglion cells of the muscle spindle afferents from the jaw-closing muscles are located. In contrast, no such change occurred after blocking periodontal afferents by transection of both the maxillary and the inferior alveolar nerves. The putative feed-forward control of the FMR is therefore dependent mainly on sensory inputs from the muscle spindles, but little on those from the periodontal receptors, if any. We further examined the involvement of the CMA with the putative feed-forward control of the FMR via the transcortical loop. For this purpose, rhythmical jaw movements were induced by stimulation of the pyramidal tract. No significant change in the timing of the FMR occurred after the CMA ablation, which strongly suggests that the CMA is not involved in the putative feed-forward control of the FMR. The FMR was also noted to increase significantly in a hardness-dependent manner even after the MesV lesion, although the rate of increment decreased significantly. Contribution of muscle spindles and periodontal receptors to the hardness-dependent change of the FMR is discussed.

[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.

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.

[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.

Effect of beta-blocker eyedrops on corneal epithelial barrier function.

To investigate the long-term effect of a topically applied beta-blocker on human corneal epithelium, the corneal epithelial barrier function and the superficial cell area of the corneal epithelium were evaluated. Seventeen normal healthy volunteers (without medication), 7 cataract patients (treated with pyrenoxine eyedrops) and 7 glaucoma or ocular hypertension patients (treated with 0.5% timolol maleate) were assigned to this study. The eyedrops had been used on a daily basis for at least 3 months. In the evaluation of corneal epithelial barrier function, fluorescein uptake was measured using a slitlamp fluorophotometer after application of 3 microl of 0.5% fluorescein for 10 min. In the evaluation of the superficial cell area, the central corneal epithelium was measured by tandem scanning confocal microscopy (TSCM). The healthy control and timolol groups were compared. Corneal fluorescein uptake in the healthy control, pyrenoxine and timolol groups was 20.3 +/- 3.2, 21.5 +/- 4.0 and 76.2 +/- 30.0 ng/ml (mean +/- standard error), respectively. There was a significantly higher fluorescein uptake in the timolol group compared to the pyrenoxine group (p = 0.0088) and the healthy control group (p = 0.0055). TSCM showed no significant difference in the superficial cell areas of the corneal epithelium between the healthy control and timolol groups. beta-Blocker eyedrops decreased the corneal epithelial barrier function. Their application was not accompanied by any biomicroscopic change in the superficial cell area.

[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.

Association of two nuclear proteins, Npw38 and NpwBP, via the interaction between the WW domain and a novel proline-rich motif containing glycine and arginine.

We have previously reported a nuclear protein possessing a WW domain, Npw38 (Komuro, A., Saeki, M., and Kato, S. (1999) Nucleic Acids Res. 27, 1957-1965). Here we report a Npw38-binding protein, NpwBP, isolated from HeLa cell nuclear extracts and its characterization using a cloned cDNA. NpwBP contains two proline-rich regions that are capable of binding to the WW domain of Npw38. The binding analysis using an oligopeptide-immobilized membrane revealed that the WW domain of Npw38 preferentially recognizes a short proline-rich sequence, PPGPPP, surrounded by an arginine residue, so we named it a PGR motif. Localization analysis using green fluorescent protein fusion protein and immunostaining showed that Npw38 and NpwBP are colocalized in the same subnuclear region. Coimmunoprecipitation experiments confirmed the association between Npw38 and NpwBP, which were expressed as epitope-tagged forms in COS7 cells. Furthermore, the N-terminal region of NpwBP has binding ability for poly(rG) and G-rich single-stranded DNA. These results suggest that NpwBP is a physiological ligand of Npw38 and that the Npw38-NpwBP complex may function as a component of an mRNA factory in the nucleus.

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.

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.

Npw38, a novel nuclear protein possessing a WW domain capable of activating basal transcription.

We have found a novel cDNA encoding a 265 amino acid protein possessing a WW domain in our full-length cDNA bank. The WW domain was sandwiched between an acidic region and an acidic-basic amino acid repetitive region. In vitro transcription/translation of the cDNA produced a 38 kDa product that was also found in the cell lysate by western blot analysis. Thus this protein is named the nuclear protein containing a WW domain with a molecular mass of 38 kDa, Npw38. Immunofluorescence studies and expression of a fusion protein to a green fluorescent protein revealed that this protein is localized in the nucleus. Npw38 was shown to be capable of binding to a poly(rG) resin. Interestingly, the WW domain of Npw38 was found to function as a transcriptional activator in CHO cells using the GAL4 DNA-binding fusion system. Furthermore, the WW domains of human YAP and Pin1 were demonstrated to have a similar transcription-promoting activity. Combined mutation of the conserved first and second Trp residues and a hydrophobic triplet of TyrTyrTrp in the WW domain of Npw38 abolished the transcription-promoting activity, but single mutations of these sites did not. These results suggest that some WW domains potentially possess transcription-promoting activity in mammalian cells.

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.

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.