Effects of clotrimazole on the growth, morphological characteristics, and cisplatin sensitivity of human glioblastoma cells in vitro.

OBJECT: Clotrimazole, an antimycotic drug, inhibits proliferation of normal and cancer cells by downregulating the movement of intracellular Ca++ and K+. The authors examined the effect of clotrimazole on the growth and sensitivity to cisplatin of two human glioblastoma cell lines--A172, which has the wild-type p53 gene, and T98G, which has the mutant p53 gene in vitro. METHODS: The A172 and T98G glioblastoma cells were exposed to clotrimazole and cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium chloride colorimetric assay. Clotrimazole produced a dose-dependent inhibition of cell proliferation and caused changes in cellular structure toward a well-differentiated form. The growth inhibitory effect of clotrimazole was reversible. Western immunoblot analysis revealed a marked increase in cellular glial fibrillary acidic protein and wild-type p53 and a decrease in c-myc and c-fos oncoproteins in both cell lines treated with clotrimazole. Flow cytometric analysis revealed that clotrimazole-treated cells accumulated in the G0/G1 phase with a marked decrease in cells in the S phase; when clotrimazole was washed out from the culture medium, cells again started to proliferate, with a marked decrease in cells in the G0/G1 phase and an increase in cells in the S phase. The growth inhibitory effect of clotrimazole could not be overcome by exogenous stimulation with either epidermal growth factor or c-myc peptide. A combined treatment with clotrimazole and cisplatin significantly enhanced cell cytotoxicity compared with treatment using either drug alone. A DNA fragmentation assay showed that both clotrimazole and cisplatin induced apoptosis, which was increased in cells treated by both drugs. CONCLUSIONS: The present study indicates that clotrimazole inhibits cell proliferation accompanied by morphological changes toward differentiation of glioblastoma cells and that this drug synergistically enhances the antitumor effect of cisplatin by inducing wild-type p53-mediated apoptosis.

Immunohistochemical analysis of p53 and p21 in human primary glioblastomas in relation to proliferative potential and apoptosis.

The Cdk inhibitor p21/WAF1 can be transcriptionally activated by wild-type p53, not by mutant p53, and functions to block cell-cycle progression in many human neoplasms. We examined the immunohistochemical expression of p53 and p21 in 35 human primary glioblastomas in relation to tumor proliferation potential as assessed by the Ki-67 labeling index (LI) and the glioblastoma apoptosis index (AI). The expression of mutant p53 was observed in 74% of glioblastomas, wild-type p53 in 18% of glioblastomas, and p21 in 57% of glioblastomas. p21 expression was seen in 15 of 26 mutant p53-positive and 2 of 4 wild p53-positive tumors. Tumor Ki-67 LI correlated neither with p53 nor with p21 expression in glioblastomas. Apoptosis was identified in all 15 glioblastomas examined, with a mean (+/-SD) Al of 1.69+/-1.54, and correlated neither with p53 (wild or mutant) nor with p21 expression. The results of the present study suggest that p53 mutation and p21 protein expression are frequent in primary glioblastoma but lack correlation with tumor proliferation potential and apoptosis. The lack of correlation between p21 and p53 also suggests that p21 in glioblastomas may be induced by a p53-independent pathway.

Gap formation and species diversity in Japanese beech forests: a test of the intermediate disturbance hypothesis on a geographic scale.

To evaluate whether the intermediate-disturbance hypothesis applies on regional scales, the relationship between the species diversity and gap formation regime of beech forests was examined. The mean gap size and the variation of gap sizes showed no correlation with species diversity. The mean windstorm interval varied widely, but geographical trends, such as latitudinal gradient, were not observed. However, locations that sustained an intermediate frequency of disturbance had the highest species diversity. Although a latitudinal gradient of disturbance was not apparent, the intermediate-disturbance hypothesis was partly supported on a geographic scale. The most predictable model for species diversity was a multiple regression model composed of two factors, the windstorm interval and the cumulative temperature of the growing season. The fact that the temperature was of greater importance than the disturbance interval indicates that the most important factor in predicting forest species diversity is the amount of available energy on a geographic scale.

Cardiorespiratory dynamics during sinusoidal and impulse exercise in man.

Dynamic characteristics of ventilation, cardiac output, and gas exchange during sinusoidally varying work rates for the periods from 1 to 12 min and impulse work rate with a duration of 10 sec were studied on five healthy men in an upright position. Changes in work rate were given by controlling externally the electromagnetic braking system of a bicycle ergometer. Stroke volume, heart rate, and cardiac output during exercise were determined continuously by using an automated impedance cardiograph. Breath by breath determination in minute ventilation, respiratory frequency, tidal volume, oxygen consumption, carbon dioxide output, end-tidal pressures of oxygen and carbon dioxide, and gas exchange ratio were conducted. From these and steady-state response data amplitude and phase relations between each variable and the input work loads were obtained utilizing the frequency analysis techniques. The response characteristics to sinusoidal stimuli were well represented by first-order models with time constants for VE, VCO2, VO2, and Q averaging 75, 67, 52, and 36 sec, respectively. The kinetics of HR closely resembled that of Q. There was a close link between both the dynamics of VE and VCO2. On the other hand, the responses to impulse stimuli were better described by second-order models in which fast and slow response components were connected in parallel. However, the contribution of the fast component to total response was small. Although this response may support in its form the neuro-humoral concept to explain exercise hyperpnea, a tight linkage was observed between VE and VCO2 responses to impulse stimuli. Thus, hyperpnea during the unsteady-state of exercise may be explained by the cardiodynamic hypothesis.

The dynamic response of the cardiopulmonary parameters to passive head-up tilt.

Transitional changes in stroke volume, heart rate, and cardiac output resulting from head-up tilt were measured in healthy male subjects, using impedance cardiography. Respiratory frequency, tidal volume, ventilation rate, end-tidal O2 and Co2 tensions, O2 uptake, CO2 output, and exchange ratio were also determined breath-by-breath. When the subject was tilted upward from a supine to an upright position, the stroke volume was decreased while the heart rate was increased. The time required to attain the steady state was shorter in the stroke volume change than in the heart rate. In addition, since the heart rate was not increased so high as to be inversely related to the stroke volume, the cardiac output decreased by tilting upward. The ventilation rate remained almost unchanged within a tilt angle of 60 degrees or so. Thus, both the O2 and CO2 fractions in expired air showed transitory changes. As the original body position was restored, the cardiac output was increased to the initial level, the tidal volume showing a transient increase. During the recovery process, PCO2, in expired air increased and PO2 decreased, showing that the change in cardiac output exceeded that in ventilation in quantity. The change in ventilation moreover, preceded that of expired PCO2 by ca. 10 sec, suggesting that the enhancement of ventilation occurred through some agent(s) other than the chemical drive.

Infrared spectra of 1 -monopalmitin- or 1-monostearin-water systems in the gel phase.

The infrared spectra of 1-monopalmitin- or 1-monostearin-water systems in the gel phase were observed at room temperature. In both systems the infrared intensities of the bands parallel and perpendicular to the paraffin chain are relatively reduced and enhanced, respectively, on going from the crystalline phase to the gel phase. These spectral changes are explained in terms of the interaction among oscillating dipoles, which is sensitive to the morphology change from the three-dimensional crystalline phase to the two-dimensional lipid bilayers. The non-planar lipid bilayers are proposed for the gel phase in monopalmitin-water systems with x > or = 35 (x: wt.% water).

Asymptomatic brain tumor detected at brain check-up.

Brain check-up was performed in 4000 healthy subjects who underwent medical and radiological examinations for possible brain diseases in our hospital from April 1996 to March 2000. Magnetic resonance imaging revealed 11 brain tumors which consisted of six meningiomas, three pituitary adenomas, one astrocytoma, and one epidermoid cyst. The detection rate of incidental brain tumor in our hospital was 0.3%. Nine patients underwent surgery, with one case of morbidity due to postoperative transient oculomotor nerve paresis. The widespread use of brain check-up may increasingly detect asymptomatic brain tumors. Surgical indications for such lesions remain unclear, and the strategy for treatment should be determined with consideration of the patient's wishes.

[Cerebral arteritis and cerebritis caused by subdural empyema: two cases report].

We report two cases of cerebral angitis and cerebritis caused by subdural empyema. A 22-year-old man, who complained of a headache and high fever, suddenly developed unconsciousness and right hemiparesis. CT and MRI demonstrated left subdural empyema with diffuse brain swelling. CT angiography showed diffuse narrowing of the left internal carotid artery, middle cerebral artery, and anterior cerebral artery. Although we performed craniotomy, continuous irrigation with drainage, systemic injection of antibiotics for subdural empyema, antiplatelet therapy, and hyperbaric oxygen therapy for angitis, his condition remained unchanged. A 67-year-old man who had previously undergone burr hole surgery presented to our hospital for the treatment of scalp infection. He suddenly developed unconsciousness and right hemiparesis. CT and MRI demonstrated left subdural empyema with diffuse brain swelling, but MR angiography did not show any abnormal findings. Hemiparesis improved after the surgery and systemic injection of the antibiotics. Subdural empyema with sinusitis or meningitis around the skull base sometimes causes cerebral angitis. We considered that the angiographical evaluation for the subdural empyema was necessary to detect angitis.

[Preoperative OMF therapy for gastric cancer--examination of macroscopical effect in comparison with histological effect].

OMF treatment consisting of perifocal administration of OK-432 associated with treatment with MMC and 5-FU was applied for 14 lesions of 13 cases including 1 case of multiple cancer. The results obtained were examined macroscopically and histologically. Macroscopic findings were rated as "good" in 5 lesions (35.8%), "fair" in 8 lesions (57.1%) and "poor" in 1 case. There was no significant difference according to a histologic type. Histologic efficacy was assessed as "++" for 6 lesions (43%), "+" for 6 lesions (43%) and "-" for 2 lesions (14%), suggesting a tendency that higher histologic efficacy be obtained for lesions with a lower degree of differentiation. Taking "+" or higher histologically as "effective", the efficacy rate was approximately 86%. When the macroscopic results were compared with histologic results based on a criteria for assessment established by our group, it was found that the macroscopic efficacy tended to be associated with the histologic efficacy with a consistency rate of approximately 71%.

[Preoperative immunochemotherapy for gastric cancer combined with perifocal administration of OK-432].

OMF treatment consisting of perifocal administration of OK-432 associated with MMC and 5-FU was applied for 37 lesions of 35 cases including 2 with multiple cancer. The results obtained were as follows lesions. Macroscopical efficacy was rated as "good" in 12 lesions (32.4%), "fair" in 24 lesions (64.9%) and "poor" in 1 case. Histological efficacy was assessed as "++" for 10 lesions (27%), and "+" for 16 lesions (43.2%). Taking "+" or higher histologically as "effective", the efficacy rate was approximately 71%. Efficacy rate for metastatic lymph nodes was approximately 61% in patients who underwent a curative operation. Patients who received non-curative operation showed a certain efficacy to some extent. Twenty-one cases were judged as "S2" at operation. 11 cases as "se", and another 10 cases as 2 cases of "ss beta" and 8 cases of "pm" Histologically . Seven cases of "pm" were considered either as "good" or Histological efficacy as "++" in terms of macroscopical efficacy.

[Peri-focal administration of OK-432 combined with radiotherapy in the treatment of esophageal cancer].

We have been interested in direct activity of OK-432 on cancer lesion and applied a combined therapy of perifocal administration of OK-432 with radiation for 2 cases as a new trial. Following results were obtained. Histological efficacy as Ef3 at resected specimen was observed in one case and complete regression of cancer lesion was confirmed by X-ray, fiber scope and biopsy after combined therapy of perifocal administration of 10 KE of OK-432 with radiation of 2400 rad in total.

Costs and benefits of photosynthetic light acclimation by tree seedlings in response to gap formation.

Some shade leaves increase their photosynthetic capacity (P (max)) when exposed to a higher irradiance. The increase in P (max) is associated with an increase in chloroplast size or number. To accommodate those chloroplasts, plants need to make thick leaves in advance. We studied the cost and benefit of photosynthetic acclimation in mature leaves of a tree species, Kalopanax pictus Nakai, in a cool-temperate deciduous forest. Costs were evaluated as the additional investment in biomass required to make thick leaves, while the benefit was evaluated as an increase in photosynthetic carbon gain. We created gaps by felling canopy trees and examined the photosynthetic responses of mature leaves of the understorey seedlings. In the shade, leaves of K. pictus had vacant spaces that were not filled by chloroplasts in the mesophyll cells facing the intercellular space. When those leaves were exposed to higher irradiance after gap formation, the area of the mesophyll surface covered by chloroplasts increased by 17% and P (max) by 27%. This increase in P (max) led to an 11% increase in daily carbon gain, which was greater than the amount of biomass additionally invested to construct thicker leaves. We conclude that the capacity of a plant to acclimate to light (photosynthetic acclimation) would contribute to rapid growth in response to gap formation.

Generation of anti-tumour effector T cells from naïve T cells by stimulation with dendritic/tumour fusion cells.

Tumour-draining lymph node T cells are an excellent source of effector T cells that can be used in adoptive tumour immunotherapy because they have already been sensitized to tumour-associated antigens in vivo. However, such tumour-specific immune cells are not readily obtained from the host due to poor immunogenicity of tumours and reduced host immune responses. One obstacle in implementation of adoptive immunotherapy has been insufficient sensitization and expansion of tumour-specific effector cells. In this study, we aim to improve adoptive immunotherapy by generating anti-tumour effector T cells from naïve T lymphocytes. We attempted to achieve this by harnessing the advantages of dendritic cell (DC)-based anti-cancer vaccine strategies. Electrofusion was routinely employed to produce fusion cells with 30-40% efficiency by using the poorly immunogenic murine B16/F10 cell line, D5 cells, and DC generated from bone marrow cells. CD62L-positive T cells from spleens of naïve mice and the fusion cells were cocultured with a low concentration of IL-2. After 9 days of culture, the antigen-specific T cells were identified with an upregulation of CD25 and CD69 expression and a downregulation of CD62L expression. These cells secreted IFN-gamma upon stimulation with irradiated tumour cells. Moreover, when transferred into mice with 3-day established pulmonary metastases, these cells with coadministration of IL-2 exhibited anti-tumour efficacy. In contrast, naïve T cells cocultured with a mixture of unfused DC and irradiated tumour cells did not exhibit anti-tumour efficacy. Our strategy provides the basis for a new approach in adoptive T cell immunotherapy for cancer.